Methods of modulating functions of polypeptide GalNAc-transferases and of screening test substances to find agents herefor, pharmaceutical compositions comprising such agents and the use of such agents for preparing medicaments

ABSTRACT

Attachment of O-glycans to proteins is controlled by a large family of homologous polypeptide GalNAc-transferases. Polypeptide GalNAc-transferases contain a C-terminal sequence with similarity to lectins. This invention discloses that the putative lectin domains of GalNAc-transferase isoforms, GalNAc-T4, -T7, -T2, and -T3, are functional and recognize carbohydrates, glycopeptides, and peptides and discloses the lectin domains of GalNAc-T1-T16. These lectin domains have different binding specificities and modulate the functions of GalNAc-transferase isoforms differently. Novel methods for identification of inhibitors or modulators of binding activities mediated by lectin domains of polypeptide GalNAc-transferases are disclosed. Direct binding activity of GalNAc-transferase lectins has been demonstrated for the first time and methods to measure lectin mediated binding of isolated lectins or enzymes with lectin domains are disclosed. The present invention specifically discloses a novel selective inhibitor of polypeptide GalNAc-transferase lectin domains, which provides a major advancement in that this inhibitor and related inhibitors sharing common characteristics of activity bind lectin domains without serving as acceptor substrate for glycosyltransferases involved in synthesis of O-glycans. This inhibitor is represented by the β-anomeric configuration of GalNAc-benzyl, GalNAcβ-benzyl. Methods for inhibiting intracellular transport, cell surface expression, and secretion of mucins and O-glycosylated glycoproteins without affecting O-glycosylation processing are disclosed using the novel selective inhibitor identified.

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Serial No. TO BE ASSIGNED filed Nov. 8, 2002 andentitled “Methods to Identify Agents Modulating Functions of PolypeptideGalNAc-transferases, Pharmaceutical Compositions Comprising Such Agentsand the Use of Such Agents for Preparing Medicaments” by Henrik Clausenand Eric Paul Bennett (Attorney Docket No. 4305/OM243). This applicationis also a continuation-in-part of International Patent Application No.PCT/DK01/00328 filed May 10, 2001, which published on Nov. 15, 2001 asInternational Publication No. WO 01/85215. PCT/DK01/00328 claimspriority of U.S. Provisional Patent Application No. 60/203,331 filed onMay 11, 2000, which is now abandoned. Each of these priorityapplications is incorporated herein by reference and in their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to the biosynthesis,sorting and secretion of mucins, O-glycosylated glycoproteins, andglycoproteins. In particular, this invention concerns a method ofmodulating functions of a homologous family ofUDP-N-acetyl-α-D-galactosamine: polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases), which addN-acetylgalactosamine (GalNAc) to the hydroxy group of serine andthreonine amino acid residues in peptides and proteins.

[0003] Further, this invention concerns a method of screening one ormore test substances for the ability to modulate polypeptideGalNAc-transferase enzymatic activity in a cell-free or cell-basedassay, in order to find agents which are effective in binding to one ormore lectin domains of polypeptide GalNAc-transferases, for example,selective inhibitors of the binding properties of the above mentionedlectin domains and selective inhibitors of the effects that these lectindomains exert on intracellular transport, transport to cell surface, andsecretion of mucins, glycoproteins, and proteins.

[0004] Even further, this invention provides a preferable inhibitor,GalNAcβ-benzyl, which is a novel inhibitor and representative of a novelgroup of inhibitors which display the common characteristic ofselectively inhibiting lectins of polypeptide GalNAc-transferases indirect binding assays and not serve as substrates for otherglycosyltransferases involved in O-glycan biosyntheses, while exhibitinginhibitory activity of secretion and intracellular transport of mucinsand glycoproteins in cells. GalNAcβ-benzyl and related inhibitors withthe same biological functions represent preferable selective inhibitorcompared to GalNAcα-benzyl because these do not serve as substrates forglycosyltransferases extending O-glycans and do not provide a generalinhibition of the O-glycosylation process in cells.

[0005] Also, the present invention concerns pharmaceutical compositionscomprising an agent which is effective in modulating functions of one ormore polypeptide GalNAc-transferases, as well as the use of an agentwhich is effective in inhibiting one or more lectin domains ofpolypeptide GalNAc-transferases and modulating functions mediated bysaid lectin domains for preparing medicaments for the treatment ofvarious disorders.

BACKGROUND OF THE INVENTION

[0006] Mucin-type O-glycosylation, one of the most abundant forms ofprotein glycosylation, is found on secreted and cell surface associatedglycoproteins of all eukaryotic cells except yeast. Mucin-type O-glycanscontribute to a number of important molecular functions, including:direct effects on protein conformation, solubility, and stability;specific receptor functions that regulate cell trafficking and cell-cellinteractions; and microbial clearance. Mucin-type O-glycans aresynthesised in the Golgi through the sequential addition of saccharideresidues, first to hydroxyl groups on serines and threonines of aprotein core and subsequently to hydroxyl groups on the growingsaccharide chains that extend from the protein core. There is greatdiversity in the structures created by O-glycosylation (hundreds ofpotential structures), which are produced by the catalytic activity ofhundreds of glycosyltransferase enzymes that are resident in the Golgicomplex. Diversity exists at the level of the glycan structure and inpositions of attachment of O-glycans to protein backbones. Despite thehigh degree of potential diversity, it is clear that O-glycosylation isa highly regulated process that shows a high degree of conservationamong multicellular organisms.

[0007] The factors that regulate the attachment of O-glycans toparticular protein sites and their extension into specific structuresare poorly understood. Longstanding hypotheses in this area propose thatmucin-type O-glycosylation occurs in a stochastic manner where structureof acceptor proteins combined with topology and kinetic properties ofresident Golgi glycosyltransferases determine the order and degree ofglycosylation (1). This concept does not fully explain the high degreeof regulation and specialisation that governs the O-glycosylationprocess. In particular it is difficult to envision how large mucinmolecules with high densities of O-glycans are glycosylated in the Golgiby stochastic mechanisms that also create other sparsely glycosylatedproteins.

[0008] The first step in mucin-type O-glycosylation is catalysed by oneor more members of a large family of UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41),which transfer GalNAc to serine and threonine acceptor sites⁴².To datetwelve members of the mammalian GalNAc-transferase family have beenidentified and characterized, and several additional putative members ofthis gene family have been predicted from analysis of genome databases.The GalNAc-transferase isoforms have different kinetic properties andshow differential expression patterns temporally and spatially,suggesting that they have distinct biological functions⁴². Sequenceanalysis of GalNAc-transferases have led to the hypothesis that theseenzymes contain two distinct subunits: a central catalytic unit, and aC-terminal unit with sequence similarity to the plant lectin ricin,designated the “lectin domain” (3-6). Previous experiments involvingsite-specific mutagenesis of selected conserved residues confirmed thatmutations in the catalytic domain eliminated catalytic activity. Incontrast, mutations in the “lectin domain” had no significant effects oncatalytic activity of the GalNAc-transferase isoform, GalNAc-T1 (3).Thus, the C-terminal “lectin domain” was believed not to be functionaland not to play roles for the enzymatic functions of GalNAc-transferases(3).

[0009] Recent evidence demonstrates that some GalNAc-transferasesexhibit unique activities with partially GalNAc-glycosylatedglycopeptides. The catalytic actions of at least threeGalNAc-transferase isoforms, GalNAc-T4, -T7, and -T10, selectively acton glycopeptides corresponding to mucin tandem repeat domains where onlysome of the clustered potential glycosylation sites have been GalNAcglycosylated by other GalNAc-transferases^(7-9, 44). GalNAc-T4 and -T7recognize different GalNAc-glycosylated peptides and catalyse transferof GalNAc to acceptor substrate sites in addition to those that werepreviously utilized. One of the functions of such GalNAc-transferaseactivities is predicted to represent a control step of the density ofO-glycan occupancy in mucins and mucin-like glycoproteins with highdensity of O-glycosylation. It was hypothesized that such sequentialactions of multiple GalNAc-transferase isoforms may be required tocomplete O-glycan attachments to some mucin peptide sequences allowingfor detailed control of density.

[0010] One example of this is the glycosylation of the cancer-associatedmucin MUC1. MUC1 contains a tandem repeat O-glycosylated region of 20residues (HGVTSAPDTRPAPGSTAPPA) with five potential O-glycosylationsites. GalNAc-T1, -T2, and -T3 can initiate glycosylation of the MUC1tandem repeat and incorporate at only three sites (HGVTSAPDTRPAPGSTAPPA,GalNAc attachment sites underlined). GalNAc-T4 is unique in that it isthe only GalNAc-transferase isoform identified so far that can completethe O-glycan attachment to all five acceptor sites in the 20 amino acidtandem repeat sequence of the breast cancer associated mucin, MUC1.GalNAc-T4 transfers GalNAc to at least two sites not used by otherGalNAc-transferase isoforms on the GalNAc4TAP24 glycopeptide(TAPPAHGVTSAPDTRPAPGSTAPP, GalNAc attachment sites underlined) (8). Anactivity such as that exhibited by GalNAc-T4 appears to be required forproduction of the glycoform of MUC1 expressed by cancer cells where allpotential sites are glycosylated (10). Normal MUC1 from lactatingmammary glands has approximately 2.6 O-glycans per repeat (11) and MUC1derived from the cancer cell line T47D has 4.8 O-glycans per repeat(10). The cancer-associated form of MUC1 is therefore associated withhigher density of O-glycan occupancy and this is accomplished by aGalNAc-transferase activity identical to or similar to that ofGalNAc-T4.

[0011] The specific mechanism by which GalNAc-T4, -T7, and -T10recognize and function with GalNAc-glycosylated glycopeptides is notknown. However, it was originally demonstrated that theGalNAc-glycopeptide specificity exerted by GalNAc-T4 is directed or atleast dependent on its lectin domain. A single amino acid substitutionin the T4 lectin domain predicted to inactivate its function abolishedthe GalNAc-glycopeptide specificity of T4 without adversely affectingthe basic catalytic mechanism of the transferase⁴². This suggests thatthe lectin domain interacts with GalNAc-glycopeptides and confers anovel catalytic function to the enzyme protein. Despite extensiveattempts it has in the past not been possible to demonstrate actualbinding of the transferase and lectin to sugars and glycopeptides, butit was possible to demonstrate selective inhibition of theGalNAc-glycopeptide activity of GalNAc-T4 using 230 mM concentration ofGalNAc⁴². Millimolar concentrations of GalNAcα-benzyl can inhibit thelectin mediated GalNAc-glycopeptide substrate specificity of GalNAc-T4as well as -T7.Polypeptide GalNAc-transferases, which have not displayedapparent GalNAc-glycopeptide specificities, also appear to be modulatedby their lectin domains. Recently, it was found that mutations in theGalNAc-T1 lectin domain, similarly to those previously analysed inGalNAc-T4⁴², modified the activity of the enzyme in a similar fashion asGalNAc-T4. Thus, while wild type GalNAc-T1 added multiple consequtiveGalNAc residues to a peptide substrate with multiple acceptor sites,mutated GalNAc-T1 failed to add more than one GalNAc residue to the samesubstrate⁴⁵. The mechanism is however not understood.

[0012] Glycosylation confers physico-chemical properties includingprotease resistance, solubility, and stability to proteins (12-14).Glycosylation furthermore confers changes in immunological responses toproteins and glycoproteins. O-glycosylation on mucins and mucin-likeglycoproteins protect these molecules found in the extracellular spaceand body fluids from degradation. Control of O-glycosylation withrespect to sites and number (density) of O-glycan attachments toproteins as well as control of the O-glycan structures made at specificsites or in general on glycoproteins, is of interest for severalpurposes. Diseased cells e.g. cancer cells often dramatically changetheir O-glycosylation and the altered glycans and glycoproteins mayconstitute targets for therapeutic and diagnostic measures (15; 16).Mucins functioning in body fluids may have different propertiesdepending on density and structure of O-glycans attached in protectionagainst disease, including infections by micro-organisms. Furthermore,mucins with different glycosylation may change physico-chemicalproperties including stability and solubility properties that mayinfluence turnover and removal of mucous. A number of lung diseases,e.g. cystic fibrosis, asthma, chronic bronchitis, smokers lungs, areassociated with symptomatic mucous accumulation (17-19), and it islikely that the nature and structure of mucins play a role in thepathogenesis of such diseases.

[0013] Partial inhibitors of O-glycosylation in cells have beenreported. Aryl-N-acetyl-α-galactosaminides such as benzyl-, phenyl-, andp-nitrophenyl-GalNAc were originally found to inhibit the second step inO-glycosylation, the O-glycan processing step, by inhibiting synthesisof core 1 (Galβ1-3GalNAcα1-R) and more complex structures 20.Benzyl-αGalNAc was also found to inhibit sialylation. It is generallybelieved that the downstream effects of benzyl-αGalNAc treatment aremediated by substrate competition of biosynthetic glycosylation productsof benzyl-αGalNAc. Thus, e.g. the immediate glycosylation product ofbenzyl-αGalNAc is Galβ1-3GalNAcα-benzyl and this serves as an efficientsubstrate for the core 1 α2-3sialyltransferase ST3Gal-I^(21,22).GalNAcα-benzyl has been the most widely used inhibitor ofO-glycosylation, but it has only been used in cell culture as effectivetreatment concentrations lead to intracellular build-up of vesicles withGalNAcα-benzyl products and treated cells change morphology and growthcharacteristics⁴⁶.

[0014] Treatment of cells with benzyl-αGalNAc inhibit O-glycanprocessing and affect apical sorting of some O-glycosylatedproteins²³⁻²⁵. The mechanism for this is generally believed to bethrough inhibition of sialylation⁴⁶. Inhibition of mucin secretion hasalso been observed in culture cells, more specifically HT29 MTX cells,but this effect is not generally found in mucin secreting cells⁴⁶.

[0015] True inhibitors of O-glycosylation, i.e. inhibitors of theinitial O-glycan attachment process governed by polypeptideGalNAc-transferases have not been identified.

[0016] Inhibitors of the initiating step in O-glycosylation couldcompletely or selectively block attachment of O-glycans toO-glycosylation sites in proteins. Compounds inhibiting the catalyticfunction of a selected subset of the polypeptide GalNAc-transferasefamily may be predicted to only lead to partial inhibition ofO-glycosylation capacity of cells. Proteins with no or littleO-glycosylation may have entirely different biological properties thantheir normal glycosylated counterparts. Complete inhibition ofO-glycosylation is not desirable because of the many diverse functionsof O-glycans, and it is expected to result in cell death. Selectiveinhibition of O-glycosylation on the other hand is desirable in manycases such as cancer cells producing glycoproteins and mucins with moredense O-glycosylation than normal cells. For example breast cancer cellsappear to hyperglycosylate the cancer-associated cell surface mucin MUC1compared to glycosylation in normal cells (10). The overexpression ofMUC1 and hyperglycosylation found in cancer cells are likely to beimportant for the pathobiology of cancers. Methods of inhibiting thehyperglycosylation of mucins in cancer cells is desirable.

[0017] It is apparent from the above that inhibitors in the prior artinterfere with O-glycan processing, i.e. the glycosylation process thatextend GalNAc residues directly attached to proteins at serine andthreonine residues. Existing inhibitors of O-glycosylation are notsuitable for therapeutic treatment in mammals including man as theyprofoundly affect O-glycosylation processing as well as lead toundesired morphological and growth effects on culture cells.

[0018] Consequently, there exists a need in the art for methods ofinhibiting the functions of polypeptide GalNAc-transferases. Preferablein selectively inhibiting O-glycosylation attachments in glycoproteinsand mucins. There also exists a need in the art for therapeuticcompounds that display selectively and limited inhibition ofO-glycosylation without generally affecting the process ofO-glycosylation. The present invention meets these needs, and furtherpresents other related advantages.

SUMMARY OF THE INVENTION

[0019] Mucin-type O-glycosylation, one of the most abundant forms ofprotein glycosylation, is found on secreted and cell surface associatedglycoproteins of all eukaryotic cells except yeast. Mucin-type O-glycanscontribute to a number of important molecular functions, including:direct effects on protein conformation, solubility, and stability;specific receptor functions that regulate cell trafficking and cell-cellinteractions; and microbial clearance. Mucin-type O-glycans aresynthesised in the Golgi through the sequential addition of saccharideresidues, first to hydroxyl groups on serines and threonines of aprotein core and subsequently to hydroxyl groups on the growingsaccharide chains that extend from the protein core. There is greatdiversity in the structures created by O-glycosylation (hundreds ofpotential structures), which are produced by the catalytic activity ofhundreds of glycosyltransferase enzymes that are resident in the Golgicomplex. Diversity exists at the level of the glycan structure and inpositions of attachment of O-glycans to protein backbones. Despite thehigh degree of potential diversity, it is clear that O-glycosylation isa highly regulated process that shows a high degree of conservationamong multicellular organisms.

[0020] The factors that regulate the attachment of O-glycans toparticular protein sites and their extension into specific structuresare poorly understood. Longstanding hypotheses in this area propose thatmucin-type O-glycosylation occurs in a stochastic manner where structureof acceptor proteins combined with topology and kinetic properties ofresident Golgi glycosyltransferases determine the order and degree ofglycosylation (1). This concept does not fully explain the high degreeof regulation and specialisation that governs the O-glycosylationprocess. In particular it is difficult to envision how large mucinmolecules with high densities of O-glycans are glycosylated in the Golgiby stochastic mechanisms that also create other sparsely glycosylatedproteins.

[0021] The first step in mucin-type O-glycosylation is catalysed by oneor more members of a large family of UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41),which transfer GalNAc to serine and threonine acceptor sites⁴².To datetwelve members of the mammalian GalNAc-transferase family have beenidentified and characterized, and several additional putative members ofthis gene family have been predicted from analysis of genome databases.The GalNAc-transferase isoforms have different kinetic properties andshow differential expression patterns temporally and spatially,suggesting that they have distinct biological functions⁴². Sequenceanalysis of GalNAc-transferases have led to the hypothesis that theseenzymes contain two distinct subunits: a central catalytic unit, and aC-terminal unit with sequence similarity to the plant lectin ricin,designated the “lectin domain” (3-6). Previous experiments involvingsite-specific mutagenesis of selected conserved residues confirmed thatmutations in the catalytic domain eliminated catalytic activity. Incontrast, mutations in the “lectin domain” had no significant effects oncatalytic activity of the GalNAc-transferase isoform, GalNAc-T1 (3).Thus, the C-terminal “lectin domain” was believed not to be functionaland not to play roles for the enzymatic functions of GalNAc-transferases(3).

[0022] Recent evidence demonstrates that some GalNAc-transferasesexhibit unique activities with partially GalNAc-glycosylatedglycopeptides. The catalytic actions of at least threeGalNAc-transferase isoforms, GalNAc-T4, -T7, and -T10, selectively acton glycopeptides corresponding to mucin tandem repeat domains where onlysome of the clustered potential glycosylation sites have been GalNAcglycosylated by other GalNAc-transferases^(7-9, 44). GalNAc-T4 and -T7recognize different GalNAc-glycosylated peptides and catalyse transferof GalNAc to acceptor substrate sites in addition to those that werepreviously utilized. One of the functions of such GalNAc-transferaseactivities is predicted to represent a control step of the density ofO-glycan occupancy in mucins and mucin-like glycoproteins with highdensity of O-glycosylation. It was hypothesized that such sequentialactions of multiple GalNAc-transferase isoforms may be required tocomplete O-glycan attachments to some mucin peptide sequences allowingfor detailed control of density.

[0023] One example of this is the glycosylation of the cancer-associatedmucin MUC1. MUC1 contains a tandem repeat O-glycosylated region of 20residues (HGVTSAPDTRPAPGSTAPPA) with five potential O-glycosylationsites. GalNAc-T1, -T2, and -T3 can initiate glycosylation of the MUC1tandem repeat and incorporate at only three sites (HGVTSAPDTRPAPGSTAPPA,GalNAc attachment sites underlined). GalNAc-T4 is unique in that it isthe only GalNAc-transferase isoform identified so far that can completethe O-glycan attachment to all five acceptor sites in the 20 amino acidtandem repeat sequence of the breast cancer associated mucin, MUC1.GalNAc-T4 transfers GalNAc to at least two sites not used by otherGalNAc-transferase isoforms on the GalNAc4TAP24 glycopeptide(TAPPAHGVTSAPDTRPAPGSTAPP, GalNAc attachment sites underlined) (8). Anactivity such as that exhibited by GalNAc-T4 appears to be required forproduction of the glycoform of MUC1 expressed by cancer cells where allpotential sites are glycosylated (10). Normal MUC1 from lactatingmammary glands has approximately 2.6 O-glycans per repeat (11) and MUC1derived from the cancer cell line T47D has 4.8 O-glycans per repeat(10). The cancer-associated form of MUC1 is therefore associated withhigher density of O-glycan occupancy and this is accomplished by aGalNAc-transferase activity identical to or similar to that ofGalNAc-T4.

[0024] The specific mechanism by which GalNAc-T4, -T7, and -T10recognize and function with GalNAc-glycosylated glycopeptides is notknown. However, it was originally demonstrated that theGalNAc-glycopeptide specificity exerted by GalNAc-T4 is directed or atleast dependent on its lectin domain. A single amino acid substitutionin the T4 lectin domain predicted to inactivate its function abolishedthe GalNAc-glycopeptide specificity of T4 without adversely affectingthe basic catalytic mechanism of the transferase⁴². This suggests thatthe lectin domain interacts with GalNAc-glycopeptides and confers anovel catalytic function to the enzyme protein. Despite extensiveattempts it has in the past not been possible to demonstrate actualbinding of the transferase and lectin to sugars and glycopeptides, butit was possible to demonstrate selective inhibition of theGalNAc-glycopeptide activity of GalNAc-T4 using 230 mM concentration ofGalNAc⁴². Millimolar concentrations of GalNAcα-benzyl can inhibit thelectin mediated GalNAc-glycopeptide substrate specificity of GalNAc-T4as well as -T7.Polypeptide GalNAc-transferases, which have not displayedapparent GalNAc-glycopeptide specificities, also appear to be modulatedby their lectin domains. Recently, it was found that mutations in theGalNAc-T1 lectin domain, similarly to those previously analysed inGalNAc-T4⁴², modified the activity of the enzyme in a similar fashion asGalNAc-T4. Thus, while wild type GalNAc-T1 added multiple consequtiveGalNAc residues to a peptide substrate with multiple acceptor sites,mutated GalNAc-T1 failed to add more than one GalNAc residue to the samesubstrate⁴⁵. The mechanism is however not understood.

[0025] Glycosylation confers physico-chemical properties includingprotease resistance, solubility, and stability to proteins (12-14).Glycosylation furthermore confers changes in immunological responses toproteins and glycoproteins. O-glycosylation on mucins and mucin-likeglycoproteins protect these molecules found in the extracellular spaceand body fluids from degradation. Control of O-glycosylation withrespect to sites and number (density) of O-glycan attachments toproteins as well as control of the O-glycan structures made at specificsites or in general on glycoproteins, is of interest for severalpurposes. Diseased cells e.g. cancer cells often dramatically changetheir O-glycosylation and the altered glycans and glycoproteins mayconstitute targets for therapeutic and diagnostic measures (15; 16).Mucins functioning in body fluids may have different propertiesdepending on density and structure of O-glycans attached in protectionagainst disease, including infections by micro-organisms. Furthermore,mucins with different glycosylation may change physico-chemicalproperties including stability and solubility properties that mayinfluence turnover and removal of mucous. A number of lung diseases,e.g. cystic fibrosis, asthma, chronic bronchitis, smokers lungs, areassociated with symptomatic mucous accumulation (17-19), and it islikely that the nature and structure of mucins play a role in thepathogenesis of such diseases.

[0026] Partial inhibitors of O-glycosylation in cells have beenreported. Aryl-N-acetyl-α-galactosaminides such as benzyl-, phenyl-, andp-nitrophenyl-GalNAc were originally found to inhibit the second step inO-glycosylation, the O-glycan processing step, by inhibiting synthesisof core 1 (Galβ1-3GalNAcα1-R) and more complex structures²⁰.Benzyl-αGalNAc was also found to inhibit sialylation. It is generallybelieved that the downstream effects of benzyl-αGalNAc treatment aremediated by substrate competition of biosynthetic glycosylation productsof benzyl-αGalNAc. Thus, e.g. the immediate glycosylation product ofbenzyl-αGalNAc is Galβ1-3GalNAcα-benzyl and this serves as an efficientsubstrate for the core 1 α2-3sialyltransferase ST3Gal-I^(21,22).GalNAcα-benzyl has been the most widely used inhibitor ofO-glycosylation, but it has only been used in cell culture as effectivetreatment concentrations lead to intracellular build-up of vesicles withGalNAcα-benzyl products and treated cells change morphology and growthcharacteristics⁴⁶.

[0027] Treatment of cells with benzyl-αGalNAc inhibit O-glycanprocessing and affect apical sorting of some O-glycosylatedproteins²³⁻²⁵. The mechanism for this is generally believed to bethrough inhibition of sialylation⁴⁶. Inhibition of mucin secretion hasalso been observed in culture cells, more specifically HT29 MTX cells,but this effect is not generally found in mucin secreting cells⁴⁶.

[0028] True inhibitors of O-glycosylation, i.e. inhibitors of theinitial O-glycan attachment process governed by polypeptideGalNAc-transferases have not been identified.

[0029] Inhibitors of the initiating step in O-glycosylation couldcompletely or selectively block attachment of O-glycans toO-glycosylation sites in proteins. Compounds inhibiting the catalyticfunction of a selected subset of the polypeptide GalNAc-transferasefamily may be predicted to only lead to partial inhibition ofO-glycosylation capacity of cells. Proteins with no or littleO-glycosylation may have entirely different biological properties thantheir normal glycosylated counterparts. Complete inhibition ofO-glycosylation is not desirable because of the many diverse functionsof O-glycans, and it is expected to result in cell death. Selectiveinhibition of O-glycosylation on the other hand is desirable in manycases such as cancer cells producing glycoproteins and mucins with moredense O-glycosylation than normal cells. For example breast cancer cellsappear to hyperglycosylate the cancer-associated cell surface mucin MUC1compared to glycosylation in normal cells (10). The overexpression ofMUC1 and hyperglycosylation found in cancer cells are likely to beimportant for the pathobiology of cancers. Methods of inhibiting thehyperglycosylation of mucins in cancer cells is desirable.

[0030] It is apparent from the above that inhibitors in the prior artinterfere with O-glycan processing, i.e. the glycosylation process thatextend GalNAc residues directly attached to proteins at serine andthreonine residues. Existing inhibitors of O-glycosylation are notsuitable for therapeutic treatment in mammals including man as theyprofoundly affect O-glycosylation processing as well as lead toundesired morphological and growth effects on culture cells.

[0031] Consequently, there exists a need in the art for methods ofinhibiting the functions of polypeptide GalNAc-transferases. Preferablein selectively inhibiting O-glycosylation attachments in glycoproteinsand mucins. There also exists a need in the art for therapeuticcompounds that display selectively and limited inhibition ofO-glycosylation without generally affecting the process ofO-glycosylation. The present invention meets these needs, and furtherpresents other related advantages.

[0032] (i) contacting a polypeptide GalNAc-transferase, or a cell thatrecombinantly expresses a polypeptide GalNAc-transferase, with one ormore test substances under assay conditions suitable for the detectionof said enzymatic activity; and

[0033] (ii) measuring whether said enzymatic activity is therebymodulated by one or more of the test substances.Brief Description of theDrawings.

[0034] (iii) The present invention provides a novel method for largescale screening of test substances for the ability to inhibitlectin-mediated activity of polypeptide GalNAc-transferases in acell-free assay, which comprises:contacting an isolated polypeptideGalNAc-transferase, an isolated lectin domain from a polypeptideGalNAc-transferase, or a fragment of a polypeptide GalNAc-transferasecapable of diplaying lectin-mediated binding on its substrate, with oneor more test substances under assay conditions suitable for thedetection of said binding ability; and

[0035] (iv) measuring whether said lectin-mediated activity is therebyinhibited or modulated by one or more of the substances.

DETAILED DESCRIPTION OF AN EXEMPLARY EMBODIMENT OF THE INVENTION

[0036] The present invention also provides a method of screening one ormore test substances for the ability to inhibit or modulateintracellular transport and/or cell surface expression of mucins,O-glycosylated glycoproteins, glycoproteins and proteins in a cell-basedassay, which comprises:

[0037] (i) contacting a cell that expresses mucins, O-glycosylatedglycoproteins, glycoproteins and proteins, with one or more testsubstances under assay conditions suitable for the detection ofinhibition or modulation of said expression; and

[0038] (ii) measuring whether intracellular transport and cell surfaceexpression of said mucins, O-glycosylated glycoproteins, glycoproteinsand proteins are thereby inhibited or modulated by one or more of thesubstances.

[0039] The present invention also provides a method of screening one ormore test substances for the ability to inhibit or modulate secretionsof mucins, O-glycosylated glycoproteins, glycoproteins and proteins in acell-based assay, which comprises:

[0040] (i) contacting a cell that secretes mucins, O-glycosylatedglycoproteins, glycoproteins with one or more test substances underassay conditions suitable for the detection of inhibition or modulationof said secretion; and

[0041] (ii) measuring whether secretion of said mucins, O-glycosylatedglycoproteins, glycoproteins and proteins are thereby inhibited ormodulated by one or more of the substances.

[0042] Substances identified as agents which are effective in inhibitingone or more lectin domains of polypeptide GalNAc-transferases may e.g.be selected from the group consisting of naturally or non-naturallyoccurring carbohydrates, peptides, glycopeptides, glycoconjugates andportions and fragments thereof. They may also be found among nucleicacids as well as small organic or inorganic molecules. They include butare not limited to peptides such as soluble peptides including Ig-tailedfusion peptides, members of random peptide libraries and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids, phosphopeptides (including members of random or partiallydegenerate, directed phosphopeptide libraries), antibodies [e.g.polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, singlechain antibodies, fragments, (e.g. Fab, F(ab)2, and Fab expressionlibrary fragments, and epitope-binding fragments thereof)], andpolypeptides. A substance to be used as an agent according to theinvention may be an endogenous physiological compound or it may be anatural or synthetic compound.

[0043] Agents in accordance with the present invention are useful forchanging the density and sites of O-glycan occupancy in mucins andO-linked glycoproteins. Further uses are in changing Golgi-transport andintracellular sorting events conferred by the lectin domains ofGalNAc-transferases. For example, inhibitors of lectin domains ofGalNAc-transferases may be useful in manipulating disease-associatedO-glycosylation to augment immunity and to prepare vaccines. Further usemay be found in manipulating mucin secretion and O-glycan density indiseases associated with mucous accumulation to decrease secretion andenhance clearance of mucins. Further use may entail modulatingO-glycosylation of recombinant glycoproteins by inhibition ofpolypeptide GalNAc-transferases in host expression cells. These andother aspects of the present invention will become evident uponreference to the following detailed description and drawings.

[0044] Accordingly, the present invention also provides a pharmaceuticalcomposition comprising an agent which is effective in modulatingfunctions of one or more polypeptide GalNAc-transferases, and apharmaceutically acceptable carrier. More specifically, said agent maybe an agent which is effective in inhibiting one or more lectin domainsof polypeptide GalNAc-transferases and modulating functions mediated bysaid lectin domains and, in particular, said agent may be selected fromthe group consisting of carbohydrates, peptides, glycopeptides,glycoconjugates and portions and fragments thereof.

[0045] Further, the present invention covers the use of an agent whichis effective in inhibiting one or more lectin domains of polypeptideGalNAc-transferases and modulating functions mediated by said lectindomains for preparing a medicament for the treatment of tumors andcancers; a medicament for the treatment of lung diseases associated withmucous accumulation such as asthma, chronic bronchitis, smoker's lung,and cystic fibrosis; a medicament for the treatment of diseases ofexocrine glands associated with increased or decreased mucin secretionsuch as Sjøgren's syndrome and dry mouth; and a medicament for thetreatment of disorders associated with dysregulation ofselectin-mediated leukocyte trafficking such as autoimmunity, arthritis,leukemias, lymphomas, immunosuppression, sepsis, wound healing, acuteand chronic inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1 illustrates that the MUC1 glycopeptide specificity ofpolypeptide GalNAc-T4 is not directed by a specific glycoform. Panel Ais a schematic depiction of product development assays monitored bycapillary electrophoresis (CE) and/or MALDI-TOF mass spectrometry. Leftside illustrates MUC1 tandem repeat peptide glycoforms (open circlesindicate attachments of GalNAc) prepared by in vitro glycosylation withindicated GalNAc-transferase isoforms. Right side illustrates products(closed circles indicate GalNAc residues added) developed in 6 hours byGalNAc-T4. Glycopeptides were characterized by mass spectrometry. PanelB is an illustration of the reactions with TAP25V21 monitored bycapillary electrophoresis, where GalNAc-T1 and -T4 were mixed. Numbersabove peaks refer to numbers of moles of GalNAc incorporated into thepeptide.

[0047]FIG. 2 illustrates that the lectin domain of GalNAc-T4 selectivelydirects its MUC1 glycopeptide specificity. Panel A is a schematicdepiction of the domain structure of polypeptide GalNAc-transferasesmodified from Hagen et al. (3). Arrows indicate conserved cysteineresidues and the major conserved sequence motifs are shown withnumbering according to the sequence of GalNAc-T 1. Bold underlinedresidues in the catalytic domain indicate some residues required forcatalysis, whereas the two marked residues in the lectin domain are notessential for catalytic activity of GalNAc-T1 (3). A D459H mutation inthe lectin domain of GalNAc-T4 corresponds to the illustrated D444H inGalNAc-T1. Panel B is a time-course MALDI-TOF(matrix-assisted-laser-desorption-ionization time-of-flight) analysis ofthe glycosylation independent activities of wild-type GalNAc-T4^(459D)and the lectin mutant GalNAc-T4^(459H) using the unique substrate forthis enzyme isoform derived from PSGL-1 [Thr in bold is the acceptorsite (8)]. The control represents co-purified endogenous activity foundwith irrelevant expression constructs. Wild-type and mutant GalNAc-T4exhibit identical glycosylation independent activities. Panel C is atime-course MALDI-TOF analysis using the unique glycosylation dependentsubstrate GalNAc3TAP25V21 (GalNAc attachment sites bold and underlined,and the two available acceptor sites for GalNAc-T4 in bold). The mutantGalNAc-T4 is virtually inactive with the glycopeptide substrate.

[0048]FIG. 3 illustrates that the lectin domain of GalNAc-T4 functionsas a lectin and has selective specificity for GalNAc. Panel A:Inhibition of the glycosylation dependent function of GalNAc-T4 by freesugars. Time-course MALDI-TOF analysis of GalNAc-T4^(459D) in thepresence of 0.23 M free sugars, indicate selective inhibition ofactivity in the presence of GalNAc. Panel B: Time-course MALDI-TOFanalysis of the glycosylation independent functions of wild-type andmutant GalNAc-T4, show that GalNAc has no effect on the generalcatalytic function of the enzyme.

[0049]FIG. 4 is a multiple sequence alignment (ClustalW) of lectindomains derived from 16 human polypeptide GalNAc-transferases. Potitionsof conserved motifs CLD and Q×W in the α, β, and γ repeats areindicated. The numbering indicated in the margins reflects numbering ofthe analysed sequence region of each GalNAc-transferase. Conservedresidues are indicated by black box'ing.

[0050]FIGS. 5A and B are schematic representations of human solublesecreted MUC1 expression constructs used for stable transfectants of CHOcells. Panel A: IgG2A His-tag was inserted into Bsu36I/XbaI site ofMUC1FL, generating a His-tagged MUC1 construct containing the endogenousMUC1 secretion signal peptide. Panel B: Muc1FL Sau3AI insert wasinserted into the BamHI site of pcDNA-inf., generating a non-tagged MUC1construct containing the γ-interferon secretion signal peptide.

[0051]FIG. 6 is a plot of absorbance v. ligand dilution showingGalNAc-transferase binding to GalNAc-MUC1 glycopeptide. A direct bindingassay (ELISA) mediated by the lectin domain was developed and validatedwith soluble secreted GalNAc-T4 and -T2 enzyme proteins. ELISA plateswere coated with peptides or glycopeptides at 1 μg/ml, blocked with BSA,and incubated with biotinylated enzymes. After washing, bound enzymeproteins were detected with HRP-Streptavidin as described in detail inExample 8. Secreted soluble constructs of GalNAc-transferases which areenzymatically active may bind to (glyco)peptide substrates through theircatalytic units as originally described for GalNAc-T2²⁶. However,GalNAc-transferase binding to substrates by the catalytic domainrequires UDP and divalent cat-ions (binding destroyed by EDTAtreatment), in accordance with previous experience²⁶. Panel A: GalNAc-T4wild type enzyme proteins (□) and GalNAc-T2 (▪) selectively bindGalNAc-MUC1 glycopeptides, with no significant binding observed to theunglycosylated peptide (GalNAc-T4 wt (A) and GalNAcT2 (▴)). Panel B:Furthermore, the GalNAc-T4 lectin mutant did not bind to eitherglycosylated Muc1 (GalNAc-Muc1) () or non glycosylated Muc1 (Muc1) (∘),whereas GalNAc-T4 wild type binds GalNAc-Muc1 (□) but not nonglcosylatedMucl (▪). Binding was not affected by 10 mM EDTA. Soluble secretedGalNAc-T4 mutant, GalNAc-T4^(459H 42), in which the lectin domain hasbeen selectively inactivated by a single amino acid substitution, showedno binding demonstrating that the binding observed with the wild typeenzyme is mediated through the lectin domain.

[0052]FIG. 7 is a plot (absorbance at 495 v. concentration of inhibitor)showing inhibition of GalNAc-T4 lectin binding. Direct binding assayswere performed with preincubation of GalNAc-T4 with inhibitors followedby incubation of GalNAc-T4 in ELISA plates activated with GalNAc-Muc1 asdescribed in detail in Example8. GalNAcα-benzyl (▪) as well asGalNAcβ-benzyl (□) inhibit at 3-6 mM, whereas the control GlcNAcα-benzyl(▴) showed no inhibition. This demonstrates that the GalNAc-transferaselectin domains show no specificity for the anomeric configuration ofGalNAc, and identifies a novel inhibitor, GalNAcβ-benzyl, ofGalNAc-transferase lectins.

[0053] FIGS. 8 A-J are a series of photomicrographs showingimmunostaining of wild type CHO and transfected wild type CHO cells witha secreted MUC1 construct. CHOldlD/MUCsol-cloneD5 was established fromthis population. MUC1 expression in the cytoplasm of 10-20% cells isvisualized by HMFG2, SM3, and vu-4H5 antibodies. Anti-T antibody HH8reacted only after neuraminidase pretreatment and the anti-Tn antibodyreacted similarly before and after neuraminidase treatment. Thissuggests that cells grown in GalNAc alone produce mainly the Tnglycoform of MUC 1, while cells grown in Gal and GalNAc produce mainlythe sialylated T (core 1) glycoforms.

[0054]FIG. 9 is a series of SDS-PAGE Western analysis of MUC1 secretedfrom wild type CHO cells stably transfected cells with a secreted MUC1construct (CHOldlD/MUCsol-cloneD5). Neu+ indicates pretreatment ofsamples with neuraminidase as described in Examples. Cells were grown inculture medium after 24 or 48 hours analysed directly or afterneuraminidase treatment.

[0055]FIG. 10 A-X (left to right from top to bottom) is a series ofphotomicrographs showing immunostaining of CHO ldlD cells stablytransfected cells with a full coding cell surface secreted MUC1construct. CHOldlD/MUC1F-clone2 cells were grown in Optimem mediumwithout and with 1 mM GalNAc, and 1 mM GalNAc plus 0.1 mM Gal for 24-48hours. Cells were trypsinized, washed, air-dried on cover slides, andimmunostained as described in Examples with antibodies to MUC1 and T andTn carbohydrates. Reactivity was evaluated before and afterneuraminidase treatment of dried acetone fixed cells. +/−neu indicatesthat the staining was identical with or without neuraminidasepretreatment.

[0056]FIGS. 11A and B are SDS-PAGE Western analysis of MUC1 secretedfrom CHO ldlD cells stably transfected cells with a secreted MUC1construct. CHOldlD/MUCsol-cloneD5 cells were grown in the presence orabsence of sugars indicated, and samples of the culture medium analyseddirectly after 24-48 hours. Positive control (GalNAc-peptide) is a60-mer MUC1 tandem repeat GalNAc-glycopeptide glycosylated with humanpolypeptide GalNAc-transferase GalNAc-T2. Lane labeled control includesmedium from CHO ldlD cells. Anti-MUC1 monoclonal antibodies 5E5 andHMFG2 were used.

[0057]FIGS. 12A and B are SDS-PAGE Western analysis of MUC1 secretedfrom CHO ldlD cells stably transfected cells with a secreted MUC1construct. Same experiment as FIG. 11 using anti-MUC1 monoclonalantibodies VU-4H5 and VU-2G7.

[0058] FIGS. 13A-D are a series of photomicrographs (left to right fromtop to bottom of) anti-MUC1 antibody immunofluorescense staining of CHOldlD cells stably transfected with a full coding cell surface MUC1(CHOldlD/MUC1F-clone2). Cell grown in the presence of GalNAc weretreated with the O-glycosylation inhibitor GalNAcα-benzyl or controlGlcNAcα-benzyl. Cells were grown in plates and stained withoutpermeabilization as described in Example 10.

[0059]FIG. 14 is an SDS-PAGE Western analysis of GalNAcα-benzylinhibition of MUC1 expression in CHO ldlD cells stably transfected witha full coding MUC1 construct. Cells were grown for 24 hours (lanes 1-6)or 48 hours (lanes 7-12) in the presence of 1 mM GalNAc (lanes 1-3 and7-9) or 1 mM GalNAc and 0.1 mM Gal (lanes 4-6 and 10-12) to limit coreO-glycosylation to GalNAcα1-O-Ser/Thr and Galβ1-3GalNAcα1-O-Ser/Thr,respectively. Cells were further treated with 2 mM GalNAcα-benzyl (lanes1, 4, 7, 10), 2 mM GlcNAcα-benzyl (lanes 2, 5, 8, 11) or no inhibitor(lanes 3, 6, 9, 12). Cells were washed and lysed at 24 or 48 hours andthe lysates subjected to immunoprecipitation with monoclonal antibodyHMFG2, which broadly recognize MUC1 glycoforms. Immunoprecipitates wereanalysed by SDS-PAGE and western blot using HMFG2 antibody to detectMUC1 expression. Lane M indicates molecular markers with assigned mw.Lane C includes a control MUC1 180-mer tandem repeat peptide which hasbeen GalNAc-glycosylated with 3 moles GalNAc per repeat using GalNAc-T2.The sharp bands migrating at 100-200 kd are immunoglobulins indicated byIgG. At 24 hours the MUC1 glycoforms expressed by cells grown in GalNAcor Gal and GalNAc migrated similarly, indicating that synthesis ofsialylated core 1 O-glycans were time limited (lanes 1-6). At 48 hours,MUC1 glycoforms migrating as higher molecular weight species wereexpressed by cells grown in Gal and GalNAc (lanes 11-12). Treatment withGalNAcα-benzyl had no significant effect at 24 hours (lanes 1 and 4),but after 48 hours a significant reduction in MUC1 expression was foundin cells grown in GalNAc as well as in Gal and GalNAc (lanes 7 and 10).In the latter case a significant shift in migration further confirmedthat GalNAcα-benzyl also serves as an inhibitor of O-glycan extensionand reduces O-glycosylation to GalNAcα1-O-Ser/Thr. GlcNAα-benzyl servedas a control and had no effect on MUC1 expression and O-glycosylationcompared to untreated cells (lanes 8 and 11).

[0060]FIG. 15 is a SDS-PAGE Western analysis resulting from the sameexperiment as in FIG. 14, but using a novel monoclonal antibody, 5E5, tovisualize MUC1 expression. Cells were grown for 48 hours in the presenceof 1 mM GalNAc (lanes 1-3) or 1 mM GalNAc and 0.1 mM Gal (lanes 4-6) tolimit core O-glycosylation to GalNAcα1-O-Ser/Thr andGalβ1-3GalNAcα1-O-Ser/Thr, respectively. Cells were further treated with2 mM GalNAcα-benzyl (lanes 1, 4), 2 mM GlcNAcα-benzyl (lanes 2, 5) or noinhibitor (lanes 3, 6). Cells were washed and lysed at 48 hours and thelysates subjected to immunoprecipitation with monoclonal antibody HMFG2,which broadly recognize MUC1 glycoforms. Immunoprecipitates wereanalysed by SDS-PAGE and western blot using 5E5 antibody, whichselectively recognize GalNAc-glycosylated MUC1 expression and show noreactivity with unglycosylated MUC1 peptides. Lanes M and C as describedin legend to FIG. 14. Treatment with GalNAcα-benzyl produced asignificant reduction in MUC1 expression in cells grown in GalNAc aswell as in Gal and GalNAc (lanes 1 and 4). In cells grown in Gal andGalNAc (lanes 4-6) only weak expression of MUC1 was detected, buttreatment of cells with GalNAcα-benzyl still produced a marked shift inmigration to lower molecular weight migrating species

[0061]FIG. 16 is an SDS-PAGE Western analysis showing the identificationof a novel inhibitor, GalNAcβ-benzyl, which exhibits the same effect onmucin transport as GalNAcα-benzyl, but does not affect O-glycanextension and O-glycosylation in general. CHO ldlD cells stablytransfected with a full coding MUC1 construct were grown for 36 hours inthe presence of 1 mM GalNAc (lanes 1-3 and 7-9) or 1 mM GalNAc and 0.1mM Gal (lanes 4-6 and 10-12) to limit core O-glycosylation toGalNAcα1-O-Ser/Thr and Galβ1-3GalNAcα1-O-Ser/Thr, respectively. Cellswere further treated with 2 mM GalNAcα-benzyl (lanes 1, 4, 7, 10), 2 mMGalNAcβ-benzyl (lanes 2, 5, 8, 11) or 2 mM GlcNAca-benzyl (lanes 3, 6,9, 12). Cells were washed and lysed at 36 hours and the lysatessubjected to immunoprecipitation with monoclonal antibodies HMFG2 (lanes1-6) or 5E5 (lanes 7-12). Immunoprecipitates were analysed by SDS-PAGEand western blot using HMFG2 antibody. Lanes M and C as described inlegend to FIG. 9. Treatment with GalNAcβ-benzyl produced the same orbetter reduction in MUC1 expression as treatment with GalNAcα-benzyl incells grown in GalNAc as well as in Gal and GalNAc (lanes 2, 5, 8). Incells grown in Gal and GalNAc (lanes 4-6) MUC1 expression was reducedwith GalNAcβ-benzyl treatment (lane 5), but in contrast to cells treatedwith GalNAcα-benzyl (lane 4), GalNAcβ-benzyl produced no change in themigration of MUC1 demonstrating that this inhibitor does not affectO-glycosylation. The lack of immunoprecipitation of MUCI by 5E5 in cellsgrown in Gal and GalNAc (lanes 10-12) indicates that MUC1 isglycosylated with more complex structures than GalNAcα1-O-Ser/Thr asrecognized by this antibody.

[0062]FIG. 17 is an SDS-PAGE Western analysis resulting from the sameexperiment as FIG. 16, except that expression is visualized by themonoclonal antibody 5E5. Cells were grown for 36 hours in the presenceof 1 mM GalNAc (lanes 1-3 and 7-9) or 1 mM GalNAc and 0.1 mM Gal (lanes4-6 and 10-12) to limit core O-glycosylation to GalNAcα1-O-Ser/Thr andGalβ1-3-GalNAcα1-O-Ser/Thr, respectively. Cells were further treatedwith 2 mM GlcNAcα-benzyl (lanes 3, 6, 9, 12). Cells were washed andlysed at 36 hours and the lysates subjected to immunoprecipitation withmonoclonal antibodies HMFG2 (lanes 1-6) or 5E5 (lanes 7-12).Immunoprecipitates were analysed by SDS-PAGE and western blot using 5E5antibody. Lanes M and C as described in legend to FIG. 14. Treatmentwith GalNAcβ-benzyl produced the same or better reduction in MUC1expression as treatment with GalNAcα-benzyl in cells grown in GalNAc(lanes 2 and 8). The lack of immunostaining of MUC1 by 5E5 in cellsgrown in Gal and GalNAc indicates that MUC1 is glycosylated with morecomplex structures than GalNAcα1-O-Ser/Thr as recognized by thisantibody.

[0063] FIGS. 18A-O (left to right from top to bottom) is a series ofphotomicrographs showing that the main O-glycan phenotype of CHO ldlDcells grown in Gal and GalNAc is sialylated T and that the 5E5 antibodydoes not react with MUC1, with T or silaylated T glycoforms of MUC1.

DETAILED DESCRIPTION OF THE INVENTION

[0064] All patent applications, patents, and literature references citedin this specification are hereby incorporated by reference in theirentirety. In the case of conflict, the present description, includingdefinitions, is intended to control.

[0065] 1. Definitions

[0066] The terms used in this specification generally have theirordinary meanings in the art, within the context of this invention andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the compositionsand methods of the invention and how to make and use them.

[0067] As used herein, the term “about” or “approximately” means within50% of a given value, preferably within 20%, more preferably within 10%,more preferably still within 5%, and most preferably within 1% of agiven value. Alternatively, the term “about” or “approximately” meansthat a value can fall within a scientifically acceptable error range forthat type of value, which will depend on how qualitative a measurementcan be given the available tools. “About” or “approximately” may definea distribution around a mean value, rather than a single value.

[0068] Molecular Biology Definitions.

[0069] In accordance with the present invention, there may be employedconventional molecular biology, microbiology and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, for example, Sambrook, Fitsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred toherein as “Sambrook et al., 1989”); DNA Cloning: A Practical Approach,Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, APractical Guide to Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

[0070] “Nucleic acid” or “polynucleotide” as used herein refers topurine- and pyrimidine-containing polymers of any length, eitherpolyribonucleotides or polydeoxyribonucleotides or mixedpolyribo-polydeoxyribo nucleotides. This includes single- anddouble-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids,as well as “protein nucleic acids” (PNA) formed by conjugating bases toan amino acid backbone. This also includes nucleic acids containingmodified bases (see below).

[0071] “Complementary DNA or cDNA” as used herein refers to a DNAmolecule or sequence that has been enzymatically synthesised from thesequences present in an mRNA template, or a clone of such a DNAmolecule. A “DNA Construct” is a DNA molecule or a clone of such amolecule, either single- or double-stranded, which has been modified tocontain segments of DNA that are combined and juxtaposed in a mannerthat would not otherwise exist in nature. By way of non-limitingexample, a cDNA or DNA which has no introns are inserted adjacent to, orwithin, exogenous DNA sequences.

[0072] A plasmid or, more generally, a vector, is a DNA constructcontaining genetic information that may provide for its replication wheninserted into a host cell. A plasmid generally contains at least onegene sequence to be expressed in the host cell, as well as sequencesthat facilitate such gene expression, including promoters andtranscription initiation sites. It may be a linear or closed circularmolecule.

[0073] Nucleic acids are “hybridizable” to each other when at least onestrand of one nucleic acid can anneal to another nucleic acid underdefined stringency conditions. Stringency of hybridization isdetermined, e.g., by a) the temperature at which hybridization and/orwashing is performed, and b) the ionic strength and polarity (e.g.,formamide) of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two nucleic acids containsubstantially complementary sequences; depending on the stringency ofhybridization, however, mismatches may be tolerated. Typically,hybridization of two sequences at high stringency (such as, for example,in an aqueous solution of 0.5×SSC, at 65° C.) requires that thesequences exhibit some high degree of complementarity over their entiresequence. Conditions of intermediate stringency (such as, for example,an aqueous solution of 2×SSC at 65° C.) and low stringency (such as, forexample, an aqueous solution of 2×SSC at 55° C.), requirecorrespondingly less overall complementarily between the hybridisingsequences. Hybridization stringency has been defined in numerouspublication known to the skilled in the art (Meinkoth and Wahl,Anal.Biochem. 138,267-284, 1984; Maniatis et al., Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, 1989; J. Q.Zhang, Eur.J.Biochem. 239,835-841:1996; M.Friedman-Einat, General andComparative Endocrinology 115,354-363:1999: M. Szabo, J.Bacteriology,1544-1553:1995; S. Sau, J.Bacteriology, 21182126, 1996). Nucleic acidsare “hybridizable” to each other when at least one strand can anneal toanother nucleic acid under defined stringency conditions. Highstringency hybridization is defined as 42° C. over night hybridizationunder standard conditions (Maniatis et al., Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory Press, 1989), followedby 5 washes with 2×SSC, 0.1 % SDS at 42° C., once with 0.5×SSC, 0.1% SDSat 55° C. and once with 0.1×SSC, 0.1%SDS at 55° C. (1×SSC is 0.15M NaCl,0.015M Na citrate). Northern and Southern nucleic acid blottinghybridization techniques, especially for the purpose of investigatinghybridization specificity, is well known to those skilled in the fieldof the invention.

[0074] An “isolated” nucleic acid or polypeptide as used herein refersto a component that is removed from its original environment (forexample, its natural environment if it is naturally occurring). Anisolated nucleic acid or polypeptide contains less than about 50%,preferably less than about 75%, and most preferably less than about 90%,of the cellular components with which it was originally associated.

[0075] A “probe” refers to a nucleic acid that forms a hybrid structurewith a sequence in a target region due to complementarily of at leastone sequence in the probe with a sequence in the target region.

[0076] A nucleic acid that is “derived from” a designated sequencerefers to a nucleic acid sequence that corresponds to a region of thedesignated sequence. This encompasses sequences that are homologous orcomplementary to the sequence, as well as “sequence-conservativevariants” and “function-conservative variants”. Sequence-conservativevariants are those in which a change of one or more nucleotides in agiven codon position results in no alteration in the amino acid encodedat that position.

[0077] Function-conservative variants of polypeptide GalNAc-transferasesare those in which a given amino acid residue in the polypeptide hasbeen changed without altering the overall conformation and enzymaticactivity (including substrate specificity) of the native polypeptide;these changes include, but are not limited to, replacement of an aminoacid with one having similar physico-chemical properties. This includesbut is not limited to, replacement of an amino acid with one havingsimilar structural or physical properties, including polar or non-polarcharacter, size, shape and charge (see, e.g., Table A).

[0078] A “polypeptide” is a chain of chemical building blocks calledamino acids that are linked together by chemical bonds called “peptidebonds”. The term “protein” refers to polypeptides that contain the aminoacid residues encoded by a gene or by a nucleic acid molecule (e.g., anmRNA or a cDNA) transcribed from that gene either directly orindirectly. Optionally, a protein may lack certain amino acid residuesthat are encoded by a gene or by an mRNA. For example, a gene or mRNAmolecule may encode a sequence of amino acid residues on the N-terminusof a protein (i.e., a signal sequence) that is cleaved from, andtherefore may not be part of, the final protein. A protein orpolypeptide, including an enzyme, may be a “native” or “wild-type”,meaning that it occurs in nature; or it may be a “mutant”, “variant” or“modified”, meaning that it has been made, altered, derived, or is insome way different or changed from a native protein or from anothermutant.

[0079] A “mutation” means any process or mechanism resulting in a mutantprotein, enzyme, polypeptide, polynucleotide, gene, or cell. Thisincludes any mutation in which a protein, enzyme, polynucleotide, orgene sequence is altered, and any detectable change in a cell arisingfrom such a mutation. The altered protein, enzyme, polypeptide orpolynucleotide is a “mutant”, also called a “variant.” Typically, amutation occurs in a polynucleotide or gene sequence, by point mutations(substitutions), deletions, or insertions of single or multiplenucleotide residues. A mutation includes polynucleotide alterationsarising within a protein-encoding region of a gene as well asalterations in regions outside of a protein-encoding sequence, such as,but not limited to, regulatory or promoter sequences. A mutation in agene can be “silent”, i.e., not reflected in an amino acid alterationupon expression, leading to a “sequence-conservative” variant of thegene. This generally arises when one amino acid corresponds to more thanone codon. Table A outlines which amino acids correspond to whichcodon(s).

[0080] Thus, due to the degeneracy of the genetic code, anythree-nucleotide codon that encodes a GalNAc-transferase or aGalNac-transferace lectin domain polypeptides described herein is withinthe scope of the invention.

[0081] The terms “mutant” and “variant” may also be used to indicate amodified or altered gene, DNA or RNA sequence, enzyme, cell, etc., i.e.,any kind of mutant. Such changes also include changes in the promoter,ribosome binding site, etc.

[0082] As outlined above, amino acid substitutions are generally basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0083] In addition, modifications, which do not normally alter theprimary sequence of the GalNAc-transferase lectin domain polypeptides,include in vivo or in vitro chemical derivatization of polypeptides,e.g., acetylation, methylation, or carboxylation. Also included asvariant polypeptides of this invention are these polypeptides modifiedby glycosylation, e.g., those made by modifying the glycosylationpatterns of a polypeptide during its synthesis and processing or infurther processing steps; or by exposing the polypeptide to enzymeswhich affect glycosylation, such as mammalian glycosylating ordeglycosylating enzymes. Also embraced as variant polypeptides are theabove-identified mutagenized sequences, which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine. TABLE A Amino Acids, Corresponding Codons, andFunctionality/Property Amino Acid SLC DNA codons Side Chain PropertyIsoleucine I ATT,ATC,ATA Hydrophobic Leucine L CTT, CTC, CTA, CTG, TTA,TTG Hydrophobic Valine V GTT, GTC, GTA, GTG Hydrophobic Phenylalanine FTTT, TTC Aromatic side chain Methionine M ATG Sulphur group Cysteine CTGT, TGC Sulphur group Alanine A GCT, GCC, GCA, GCG Hydrophobic GlycineG GGT, GGC, GGA, GGG Hydrophobic Proline P CCT, CCC, CCA, CCG Secondaryamine Threonine T ACT, ACC, ACA, ACG Aliphatic hydroxyl Serine S TCT,TCC, TCA, TCG, AGT, AGC Aliphatic hydroxyl Tryptophan W TGG Aromaticside chain Glutamine Q CAA, CAG Amide group Asparagine N AAT, AAC Amidegroup Histidine H CAT, CAC Basic side chain Glutamic acid E GAA, GAGAcidic side chain Aspartic Acid D GAT, GAC Acidic side chain Lysine KAAA, AAG Basic side chain Arginine R CGT, CGC, CGA, CGG, AGA, Basic sidechain AGG Stop codons Stop TAA, TAG,TGA

[0084] As referred to herein, “sequence similarity” means the extent towhich nucleotide or protein sequences are related. The extent ofsimilarity between two sequences can be based on percent sequenceidentity and/or conservation. Amino acids other than those indicated asconserved may differ in a protein or enzyme so that the percent proteinor amino acid sequence similarity between any two proteins of similarfunction may vary and can be, for example, at least 70%, preferably 75%,more preferably 80%, even more preferably 85%, and most preferably atleast 90%, as determined according to an alignment scheme.

[0085] “Sequence identity” herein means the extent to which twonucleotide or amino acid sequences are invariant.

[0086] “Sequence alignment” means the process of lining up two or moresequences to achieve maximal levels of sequence identity (and, in thecase of amino acid sequences, conservation), e.g., for the purpose ofassessing the degree of sequence similarity. Numerous methods foraligning sequences and assessing similarity and/or identity are known inthe art such as, for example, the ClustalW method, the Cluster Method,wherein similarity is based on the MEGALIGN algorithm, as well asBLASTN, BLASTP, and FASTA (Lipman and Pearson, 1985; Pearson and Lipman,1988). When using all of these programs, the preferred settings arethose that result in the highest sequence similarity.

[0087] The term “host cell” means any cell of any organism that isselected, modified, transformed, grown, or used or manipulated in anyway, for the production of a substance by the cell, for example theexpression by the cell of a gene, a DNA or RNA sequence, a protein or anenzyme.

[0088] A “donor substrate” is a molecule recognised by, e.g., apolypeptide GalNAc-transferees and that contributes aN-acetylgalactosamine moiety for the transferase reaction. Forpolypeptide GalNAc-transferases, a donor substrate isUDP-N-acetylgalactosamine or with some GalNAc-transferase isoformsUDP-galactose. An “acceptor substrate” is a molecule, preferably apeptide, protein, glycopeptide, and glycoprotein, that is recognised by,e.g., a polypeptide GalNAc-transferase and that is the target for themodification catalysed by the transferase, i.e., receives thecarbohydrate moiety. For polypeptide GalNAc-transferases, acceptorsubstrates include without limitation peptides, proteins, glycopeptides,and glycoproteins.

[0089] The term “agonist” refers to a molecule that increases the amountof, or prolongs the duration of, the activity of the polypeptide. Theterm “enhancer” refers to a molecule that similarly increases the amountof, or prolongs the duration of, the activity of the polypeptide. Theterm “antagonist” refers to a molecule, which decreases the biologicalor immunological activity of the polypeptide. The term “inhibitor”similarly refers to a molecule, which decreases the biological orimmunological activity of the polypeptide. Agonists, antagonists, andinhibitors may include proteins, nucleic acids, carbohydrates, or anyother molecules that associate with a polypeptide GalNAc-transferase.

[0090] The term “agent” includes small molecules, peptide mimetics andpolypeptides. “Mimetics” of GalNAc-transferase lectin-domain inhibitorsare molecules that functionally mimic the structure or function of aGalNAc-transferase lectin-domain inhibitor. Molecular mimetics include,but are not limited to: small organic compounds; nucleic acids andnucleic acid derivatives; saccharides or oligosaccharides; peptidemimetics including peptides, proteins, and derivatives thereof, such aspeptides containing non-peptide organic moieties, synthetic peptideswhich may or may not contain amino acids and/or peptide bonds, butretain the structural and functional features of a peptide ligand;pyrrolidines; peptoids and oligopeptoids which are molecules comprisingN-substituted glycine, such as those described by Simon et al. (1992)Proc. Natl. Acad. Sci. USA 89:9367.

[0091] The human N-acetylgalactosaminyltransferase T1 gene (GALNT1) hasbeen described previously²⁶. The sequence of the GALNT1 mRNA and thesequence of the GalNAc-T1 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers X85018 and CAA59380,respectively.

[0092] The human N-acetylgalactosaminyltransferase T2 gene (GALNT2) hasbeen described previously²⁶. The sequence of the GALNT2 mRNA and thesequence of the GalNAc-T2 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers X85019 and CAA59381,respectively.

[0093] The human N-acetylgalactosaminyltransferase T3 gene (GALNT3) hasbeen described previously²⁷. The sequence of the GALNT3 mRNA and thesequence of the GalNAc-T3 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers X92689 and CAA63371,respectively. The human N-acetylgalactosaminyltransferase T4 gene(GALNT4) has been described previously⁸. The sequence of the GALNT4 mRNAand the sequence of the GalNAc-T4 polypeptide have been submitted toGenBank/EBI Data Bank and assigned accession numbers Y08564 andCAA69875, respectively.

[0094] The human N-acetylgalactosaminyltransferase T5 gene (GALNT5) hasbeen described previously⁷. The sequence of the GALNT5 mRNA and thesequence of the GalNAc-T5 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers AJ245539 and CAB65104,respectively.

[0095] The human N-acetylgalactosaminyltransferase T6 gene (GALNT6) hasbeen described previously⁴⁸. The sequence of the GALNT6 mRNA and thesequence of the GalNAc-T6 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers AJ133523 and CAB55325,respectively. The human N-acetylgalactosaminyltransferase T7 gene(GALNT7) has been described previously⁸. The sequence of the GALNT7 mRNAand the sequence of the GalNAc-T7 polypeptide have been submitted toGenBank/EBI Data Bank and assigned accession numbers AJ002744 andCAB60270, respectively.

[0096] The human N-acetylgalactosaminyltransferase T8 gene (GALNT8) hasbeen described previously⁴⁹. The sequence of the GALNT8 mRNA and thesequence of the GalNAc-T8 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers AJ271385 and CAB89199,respectively.

[0097] The human N-acetylgalactosaminyltransferase T9 gene (GALNT9) hasbeen described previously⁵⁰. The sequence of the GALNT9 mRNA and thesequence of the GalNAc-T8 polypeptide have been submitted to GenBank/EBIData Bank and assigned accession numbers AB040672 and BAB13699,respectively.

[0098] The human N-acetylgalactosaminyltransferase T1O nucleic acidsequence (GALNT10) and the sequence of the encoded GalNAc-T1Opolypeptide have been submitted to GenBank/EBI Data Bank. The nucleicacid accession number is AJ505950. The amino acid sequence is asfollows: MLAWRDGELEAETSSSLFLLAMQVWMCGGRMEDIPCSRVGHIYRKYVPYKVPAGVSLARVRTLKRVAEVWMDEYAEYIYQRRPEYRHLSAGDVA VQKKLRSS.LNCKSFKWFMTKIAWDLPKFYPPVEPPAAAWGEIRNVGTGLCADTKHGAL GSPLRLEG.CVRGRGEAAWNNMQVFTFTWREDIRPGDPQHTKKFCFDAISHTSPVTLYD CHSMKGNQ.LWKYRKDKTLYHPVSGSCMDCSESDHRIFMNTCNPSSLTQQWLFEHTNST VLEKFNRN.

[0099] The human N-acetylgalactosaminyltransferase T11 gene (GALNT11)has been described previously⁴³. The sequence of the GALNT11 mRNA andthe sequence of the GalNAc-T11 polypeptide have been submitted toGenBank/EBI Data Bank and assigned accession numbers Y12434 andCAC79625, respectively.

[0100] The human N-acetylgalactosaminyltransferase T12 nucleic acidsequence (GALNT12) and the sequence of the GalNAc-T12 polypeptide havebeen submitted to GenBank/EBI Data Bank. The nucleic acid accessionnumber is AJ132365. This sequence is disclosed herewith.

[0101] The human N-acetylgalactosaminyltransferase T13 nucleic acidsequence (GALNT13) and the sequence of the GalNAc-T13 polypeptide havebeen submitted to GenBank/EBI Data Bank. The nucleic acid accessionnumber is AR153422.

[0102] The references cited above for describing human GalNAc-T1-13 areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

[0103] Expression to produce enzymatically-active polypeptideGalNAc-transferases can be carried out in any number of conventionalexpression systems familiar to those skilled in the art. In oneembodiment, GalNAc-transferases are expressed in a secreted solubleform, which can be recovered from the culture medium. Such secretedenzymes lack the N-terminal cytoplasmic tail and transmembrane retentionsequence, and have N-terminal sequence starting in the predicted stemregion (depicted domain structures of polypeptide GalNAc-transferasesshown in FIG. 2A). The boundaries of the stem is N-terminally defined bythe hydrophobic signal sequence, while the C-terminal boundary is lessclearly defined but limited to the conserved catalytic unit of theenzymes as defined by multiple sequence alignments. For some isoformsincluding GalNAc-T2 the N-terminal sequence have been determined innaturally occurring soluble proteins derived from proteolyticcleavage²⁶. In another embodiment, host cells (e.g. CHO cells) areengineered to express full coding GalNAc-transferases and glycosylatesubstrates in vivo in host cells.

[0104] Expression to produce functional lectin domains of polypeptideGalNAc-transferases without the catalytic unit (or activity) can becarried out in any number of conventional expression systems familiar tothose skilled in the art. In one embodiment, GalNAc-transferase lectinsare expressed in a secreted soluble form, which can be recovered fromthe culture medium. Such secreted soluble forms lack the N-terminalcytoplasmic tail, transmembrane retention sequence, stem region and thecatalytic unit. The boundaries of the catalytic units and lectin domainsare defined by multiple sequence alignments and experimentation oflectin binding activity (multiple sequence alignment analysis of theC-terminal sequences polypeptide GalNAc-transferases including the mostC-terminal boundaries of the catalytic domains and the entire lectindomains shown in FIG. 4). The boundaries cannot be clearly defined butthe most C-terminal well-conserved sequence motif of the catalytic units(WYLENVYP) can be excluded from the lectin domains. Parts of or theentire catalytic domains may be included to produce functional lectindomains, and inclusion of inactivating mutations in the catalytic units(e.g. mutations in the D×H motif important for donor substrate binding,or residues important for acceptor substrate binding³) may be used toavoid additional binding activity mediated through the catalytic units.In another embodiment, host cells (e.g. CHO cells) are engineered toexpress full coding polypeptide GalNAc-transferases with or withoutmutations in their catalytic units and binding mediated through lectindomains are detremiend in vivo in host cells.

[0105] Cells stably or transiently transfected with full coding orsecreted expression constructs of mucins, mucin-like glycoproteins,O-glycosylated proteins, or proteins can be carried out by any number ofconventional methods familiar to those skilled in the art. In oneembodiment, the mucin MUC1 is expressed in a soluble form, which can berecovered from the culture medium (FIG. 5 illustrates MUC1 expressionconstructs used in this invention; the DNA sequence is available fromGenBank accession number M61170). In another embodiment, host cells(e.g. CHO or CHO ldlD cells) are engineered to express MUC1 on the cellsurface. In a preferred embodiment of the invention the cells aremammalian and more preferably, the cells are human.

[0106] Human cell lines expressing cell surface mucins or secretingmucins can be selected, cultured and treated by any number ofconventional methods familiar to those skilled in the art. In oneembodiment, mucins are expressed in a secreted soluble form withouttransmembrane retention sequence, which can be recovered from theculture medium. In another embodiment, host cells (e.g. CHO ldlD cells)are engineered to express full coding mucins on the cell surface.

[0107] 2. General Aspects of the Invention

[0108] In accordance with the screening method of the invention,enzymatically active GalNAc-transferases are contacted with an acceptorsubstrate and an N-acetylgalactosamine donor substrate, preferablyUDP-N-acetylgalactosamine, under conditions for transfer ofN-acetylgalactosamine from the donor substrate to the acceptorsubstrate, in the presence of one or more test substances. Glycosylatedacceptor substrate is then obtained in a varying degree.

[0109] Preferred acceptor substrates are proteins, peptides,glycoproteins, and glycopeptides. Particularly preferred acceptorsubstrates for GalNAc-T4 are GalNAc-glycosylated glycopeptides fromMUC1, MUC2, and MUC5AC tandem repeats or multimers of those molecules.Particularly preferred acceptor substrates for GalNAc-T7 areGalNAc-glycosylated glycopeptides from MUC2 and rat submaxillary glandmucin tandem repeats or multimers of those molecules. Particularlypreferred acceptor substrates for GalNAc-T2 are peptides from MUC1,MUC2, MUC5AC and MUC7 tandem repeats or multimers of those molecules.Particularly preferred acceptor substrates for GalNAc-T3 are peptidesfrom MUC1, MUC2, MUC5AC and MUC7 tandem repeats or multimers of thosemolecules. Transfer assays for carrying out glycosylation are familiarto those in the art, and are described in the literature cited above andin the examples provided below.

[0110] As noted, human GalNAc-T4 demonstrates unique acceptor substratespecificity. GalNAc-T4 has been found to transfer GalNAc to two sites inthe MUC1 tandem repeat sequence: Ser in GVTSA and Thr in PDTR using a24-mer glycopeptide with GalNAc residues attached at sites utilized byGalNAc-T1, -T2 and T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, wherein the GalNAcsites are underlined) (8). In an important aspect of the invention, theaction of GalNAc-T4 is dependent on prior GalNAc attachments at least atone site of the five acceptor sites in the MUC1 tandem repeat. Inanother important aspect of the invention this activity is dependent onthe lectin domain of GalNAc-T4. In yet another important embodiment ofthe invention this activity can be blocked by GalNAc and GalNAccontaining compounds such as benzyl-GalNAc.

[0111] As noted, human GalNAc-T7 demonstrates unique acceptor substratespecificity. GalNAc-T7 has only been found to transfer to acceptorsubstrates which have previously been partially GalNAc-glycosylated (7;9). A preferred acceptor substrate is derived from MUC2, MUC5AC and ratsubmaxillary gland mucin tandem repeats. In an important embodiment ofthe invention the activity of GalNAc-T7 can be blocked by GalNAc andGalNAc containing compounds such as benzyl-GalNAc, and Gal and Galcontaining compounds such as benzyl-Gal and Galβ1-3GalNAcα1-benzyl.

[0112] Human GalNAc-T2 demonstrates unique UDP-Gal donor substratespecificity with MUC2 peptide substrate (28). In an important embodimentof the invention the activity of GalNAc-T2 with UDP-Gal can be blockedby GalNAc and GalNAc containing compounds such as benzyl-GalNAc.

[0113] Human GalNAc-T3 demonstrates unique UDP-Gal donor substratespecificity with rat submaxillary gland mucin peptide substrate. In animportant embodiment of the invention the activity of GalNAc-T3 withUDP-Gal can be blocked by GalNAc and GalNAc containing compounds such asbenzyl-GalNAc. The lectin domains of some GalNAc-transferases, notablyGalNAc-T4 and -T7, are shown herewith to be important for theGalNAc-glycopeptide substrate specificities exhibited by theseGalNAc-transferase isoforms. The mechanism by which the lectin domainsexert this effect on the enzyme activities is unknown. However, becauseGalNAc and GalNAcoα-benzyl were found to selectively inhibit theseactivities it was hypothesized that the lectin domains functioned byrecognizing the sugar or glycopeptide in a lectin-like interaction⁴².Considerable efforts have been applied to demonstrate actual bindingwithout success in the past (Bennett et al. unpublished, personalcommunications).

[0114] In the present invention a direct binding assay was developedusing secreted soluble GalNAc-T4 and -T2, and chemoenzymaticallyproduced multimeric MUC1 tandem repeat GalNAc-glycopeptides (FIG. 6).Short MUCI glycopeptides of traditional length of 15-20 amino acids havefailed to provide significant binding in the same assay system, and oneimprovement leading to the success was the application of extendedmultimeric MUC1 GalNAc-glycopeptides. Binding was also found to anenzymatically GalNAc-glycosylated fusion protein expressed in E. coliand containing 30 amino acids of the MUC2 tandem repeat. Anotherimprovement was the use of biotinylation of the enzymes, which providedan improved signal compared to previous attempts with identifyingretained or bound enzyme by measuring activity. The specific activitiesof GalNAc-transferases as measured in in vitro assays are relativelylow, and in past attempts to use binding and elution of enzyme acitivitypresumably the detection level was not sufficient to detect binding. Thedeveloped assay was validated to demonstrate binding through the lectindomains by several ways: i) binding was selective forGalNAc-glycosylated glycopeptides with no significant binding tounglycosylated peptides; ii) a single amino acid substitution in thelectin domains of GalNAc-T4 (and -T2), known to selectively destroyGalNAc-glycopeptide specificity of these enzymes without affecting thecatalytic unit 42 abolished binding; iii) binding was not affected byEDTA treatment which is known to destroy catalytic activity ofGalNAc-transferases^(26, 28, 51, 52); iv) binding was selectivelyinhibited by the monosacharide GalNAc and not by other sugars. In orderto minimize the size and functional complexity of GalNAc-transferaselectins to be used as probes for binding studies, we used multiplesequence alignment analysis to predict and design suitable expressionconstructs for isolated lectin domains (FIG. 4). In the presentinvention a direct binding assay was developed using isolated lectindomains of GalNAc-T4 and -T2 with minimal size (FIG. 7). Analysis of thefine specificity of the binding by inhibition studies showed thatGalNAc-T2 and -T4 lectins exhibit restricted specificity for GalNAcstructures, and surprisingly that the anomeric configuration of theGalNAc residue is not important. Thus, both GalNAcα- and GalNAcβ-benzylinhibited binding to the same degree. The lectin Helix Pomatia (HP) wasused as a control plant lectin with known binding specificity forGalNAcα-structures. HP lectin showed a very different highly preferredbinding specificity for GalNAcα-structures.

[0115] Studies with GalNAcα-benzyl have shown that this compound iseffectively taken up by cells and used in the Golgi compartments⁴⁶. Itis also well known in the art that sugar-aryl compounds are taken up bythe cell and used in the Golgi compartments. Thus, the surprisingfinding that GalNAc-transferase lectins can be inhibited by βGalNAc(GalNAcβ-benzyl), provides a new tool to study the function ofpolypeptide GalNAc-transferases in vivo; GalNAcβ-benzyl, because it,too, will enter the cell and be used in the Golgi compartments.

[0116] Availability of a binding assay is a useful tool to identify andcharacterize inhibitors of GalNAc-transferase lectins. In accordancewith the binding assay method of the invention GalNAc-transferases arecontacted with a glycopeptide, glycoprotein, fusionprotein, or otherappropriate structure or polymer containing the sugar hapten structurerecognized by the GalNAc-transferase lectins, preferablyN-acetylgalactosamine, and the GalNAc-transferase protein or lectinbound is quantitatively measured. The GalNAc-transferases may be in theform of a secreted soluble construct as applied in this invention, andany extended or truncated construct of a GalNAc-transferase as well ashybrid fusion protein that maintains the binding properties of thelectin. The ligand may be in the form of a chemoenzymatically producedMUC1 GalNAc-glycopeptide as applied in this invention, and anyglycopeptide, glycoprotein, fusionprotein of any size or sequence, orother appropriate structure or polymer containing the sugar haptenstructure recognized by the GalNAc-transferase lectins. Synthesis andchemoenzymatic synthesis of glycopeptides are familiar to those in theart, and are described in the literature cited above and in the Examplesprovided below. The ligand sugar may be GalNAc, N-acetylgalactosamine,or any other sugar in any linkage and sequence recognized by aGalNAc-transferase lectin. The binding assay may be an enzyme-linkedsolid phase immunoadsorption assay (ELISA) as applied in the invention,and any variant assay hereof where binding to ligand can be detectedincluding without limitation radioimmunoassay (RIA), surface plasmonresonance (SPR), chemoluminescense, nuclear magnetic resonancespectroscopy (¹H-NMR), and other methods know in the art. Binding may bedetected by horse-radish-peroxidase HRP-Avidin biotin as applied in thisinvention, and any other detection system including without limitationenzyme reactions, fluorescence, radioactivity, spectroscopy,spectrometry and other methods. The GalNAc-transferases may be labelledby biotinylation as applied in this invention, and any other labellingincluding without limitation antibody tags, enzymes, fluorochromes,radioisotopes and other methods know in the art, as well as detected byantibodies, phage antibody fragments or other binding proteins. Theassay may used to characterize binding specificities ofGalNAc-transferase lectins, screen and identify inhibitors ofGalNAc-transferase lectins, and screen and identify competitive binderssuch as different GalNAc-transferase lectins and other lectins andproteins with binding properties for carbohydrates.

[0117] An in vivo model system for secretion of mucins was developed. Atruncated secreted expression construct of the human cell surface mucinMUC1 containing 32 tandem repeats (FIG. 5), was stably transfected intoCHO wild type and CHO ldlD cells⁵³. FIG. 8 illustrates intracellurexpression of soluble MUC1 in wild type CHO transfectant clone,wtCHO/MUC1sol-clone-C4, visualized by multiple monoclonal anti-MUC1antibodies. Analysis of glycosylation was performed with a panel ofantibodies with well-defined specificities for carbohydrate structures,and reactivity was mainly found with anti-T after pretreatment withneuraminidase to remove sialic acids. Weak staining with anti-Tn wasalso found in some cells. FIG. 9 illustrates western blot analysis ofsecreted MUC1 from the same cells. High molecular weight MUCI migratingwith apparent mw higher than 300 Kd is labelled by HMFG2, SM3, andVU-2G7, while all antibodies including VU-4H5 label a low molecularweight MUC1 migrating with apparent mw of 130 Kd and presumed torepresent virtually unglycosylated MUC1. Pretreatment with neuraminidasedecreased migration of the high molecular weight MUCI bands, and anti-Tantibody reactivity emerged. Stable MUC1 transfectants in CHO ldlDshowed similar patterns of reactivity when grown in Gal and GalNAc.

[0118] An in vivo model system for cell surface expression of mucins wasdeveloped. A full coding expression construct of the human cell surfacemucin MUC 1 containing 32 tandem repeats (FIG. 5), was stablytransfected into CHO wild type and CHO ldlD cells. CHO ldlD cells wereoriginally established by Krieger et al.⁵³ and found to have a defect inUDP-Gal/GalNAc epimerase that renders the cells incapable of producingUDP-Gal and UDP-GalNAc. Lack of UDP-Gal limits the synthesis of alltypes of glycoconjugates including glycosphingolipids, N-linked andO-linked glycoproteins. The synthesis of O-linked glycoproteins will bearrested at GalNAcα1-O-Ser/Thr with or without addition of α2,6 linkedsialic acid. In the absence UDP-GalNAc mainly O-linked mucin-typeglycoconjugates are affected, and essentially no glycosylation occur, asthe first sugar attached is GalNAc. The defect in CHO ldlD cells can beselectively restored by addition of 1 mM GalNAc and or 0.1 mM Gal to thegrowth medium⁵³. Addition of both sugars essentially restores normalglycosylation, whereas addition of GalNAc alone limits O-glycosylationto GalNAcα1-O-Ser/Thr with or without addition of α2,6 linked sialicacid, and also affects galactosylation of N-linked glycosylation andglycolipid biosynthesis. Altschuler et al.⁵⁴ have previously shown thatcell surface expression of MUC1 in CHO ldlD cells requires addition ofGalNAc.

[0119] Cell surface expression of MUC1 was established in stablytransfected CHO wildtype and CHO ldlD cells. MUC1 was detected at thesurface of non-permeabilized cells using monoclonal anti-MUC1 antibodies(FIG. 10). In accordance with Alschuler et al.⁵⁴ MUC1 surface expressionin CHO ldlD cells was only found in cells grown in GalNAc or Gal andGalNAc, whereas cells grown without sugars or only in Gal failed toexpress MUC1 at the surface. In agreement with the conclusion drawn byAltchuler et al.⁵⁴ surface expression of MUC1 was dependent only on thefirst step in O-glycosylation, the addition of GalNAc.

[0120] MUC1 produced in CHO ldlD cells grown without GalNAc is notaccumulated in Golgi, but degraded in lysosomes⁵⁴. This indicates thatmeasuring total MUC1 in cell lysates rather than exclusively at the cellsurface may be used as a measure of MUC1 expression. The experimentsshown in FIGS. 11-13 use immunoprecipitation of total cell lysates withanti-MUC1 antibody followed by western blot analysis with the same ordifferent anti-MUC1 antibody to quantify and characterize MUCIexpression in cells. MUC1 produced in cell grown without GalNAc or onlyin the presence of Gal migrate close to the predicted mass of theprotein core. With the addition of GalNAc to the medium high molecularweight forms of MUC1 are found, and these react with all antibodiesexcept VU-2G7. The antibody 5E5 only reacts with Tn glycoforms and lackof reactivity with MUC1 from cells grown in Gal and GalNAc indicate thatthe majority of MUC1 produced is glycosylated with sialyl-T structures.

[0121] GalNAcα-benzyl is a well-known inhibitor of O-glycosylationextension⁴⁶. Treatment of cells with 1-2 mM GalNAcα-benzyl partiallyblocks core 1 O-glycosylation including α2,3 sialylation. Treatment ofcells with GalNAcα-benzyl is also known to affect surface expression ofmucins and O-glycosylated glycoproteins, as well as in some casessecretion of mucins. A number of mammalian cell lines have been treatedwith 1-2 mM GalNAcα-benzyl in the past and the resulting effects onO-glycosylation as well as mucin secretion have varied with cell type(for a detailed review see⁵⁵)

[0122] The effect of GalNAcα-benzyl on mucin transport and secretion hasbeen concluded to be due to blockage of O-glycosylation extension⁵⁵.FIGS. 13-15 illustrate that CHO ldlD cells grown in Gal and GalNAc (orwild type CHO cells), and treated with 2 mM GalNAcα-benzyl in agreementwith this, exhibits reduced expression of MUC1 as well as alteredO-glycosylation as judged by an altered SDS-PAGE migration pattern.Wildtype CHO cells as well as CHO ldlD cells grown in Gal and GalNAcproduce O-glycans of the mono- and disialylated core 1 structures(NeuAcα2-3Galβ1-3[NeuAcα2-6]+/−GalNAcα-O-Ser/Thr). Treatment withGalNAcα-benzyl results in some exposure of unsialylated core 1 asevaluated by staining with anti-T monoclonal antibody HH8, whereas onlyvery little Tn is exposed as evaluated with anti-Tn monoclonal antibody5F4. The altered SDS-PAGE migration of MUC1 produced in CHO ldlD cellsgrown in Gal and GalNAc (or wild type CHO cells, not shown) shown inFIGS. 14-15 (lanes 4 and 10 when indicated) reflects mainly loss ofsialic acids.

[0123] If the effect of GalNAcα-benzyl treatment on mucin transport andsecretion is due to inhibition of sialylation, then treatment of CHOldlD cells grown only in GalNAc and hence producing onlyGalNAcα1-O-Ser/Thr O-glycosylation (neglible STn is produced asevidenced by lack of staining with anti-STn monoclonal antibodies 3F1and TKH2, while cells stain very strongly with anti-Tn monoclonalantibodies 5F4 and 1E3), should have no effect on expression of MUC1 inthese cells. Surprisingly as shown in FIGS. 14-15 (lanes 1 and 7 whenindicated), GalNAcα-benzyl treatment does inhibit MUC1 expression in CHOldlD cells with O-glycosylation controlled and limited to Tn glycoforms.This result shows for the first time that mucin transport and secretionmay be directly affected by treatment with GalNAcα-benzyl and notthrough a mechanism involving inhibtion of sialylation or theO-glycosylation extension pathways. Combined with the findings ofAltchuler et al.⁵⁴, these results indicate that mucin transport andsecretion requires some degree of GalNAc O-glycosylation, whereasO-glycan extension including sialylation seems to be of less importancefor this process.

[0124] An appropriate control for GalNAcα-benzyl treatment has notpreviously been studied. Selection of a benzyl monosaccharide that isnot involved in and does not affect glycosylation pathways in cells isproblematic. We chose to use GlcNAca-benzyl as such a control as thisstructure is not used in glycosylation pathways of mammalianglycoproteins and glycosphingolipids. As shown in FIGS. 14-15 (lanes2,5, 8, and 11, when indicated) treatment of CHO ldlD cells with 2 mMGlcNAca-benzyl had no effect on MUC1 expression and glycosylation.GlcNAcα-benzyl thus serves as a control for treatment of cells withbenzyl sugars, and this is important because benzyl sugars and theirbiosynthetic products appear to aggregate in cells and causemorphological changes with prolonged treatment⁴⁶.

[0125] Since transport of mucin in cells was selectively inhibited byGalNAcα-benzyl (and not GlcNAcα-benzyl), even in cells limited toGalNAcα1-O-Ser/Thr O-glycosylation, we hypothesized that polypeptideGalNAc-transferases and in particular their lectin domains could beinvolved in ensuring mucin transport and preventing direction tolysosomes. One hypothesis would suggest that inhibition of theGalNAc-glycopeptide acceptor substrate specificity ofGalNAc-transferases leads to mucin glycoforms with lower density ofO-glycan occupancy (shown in vitro for e.g. MUC1 tandem repeats⁴²), andthat this decrease in O-glycan density results in increased targeting tolysosomal degradation and hence decrease in expression. Anotherhypothesis would suggest that the lectin domains of GalNAc-transferasesin general have the capacity to bind GalNAc and hence provide a lectinmediated chaperone-like function, which is required for Golgi transportof O-glycosylated proteins. Lectin chaperones are well known to functionER transport as well as in lysosomal targeting⁵⁶, but the existence ofsuch lectin chaperones for cell surface expression and secretion havenot been demonstrated in the Golgi or trans-Golgi network.

[0126] As described above we found in the present invention thatGalNAc-transferase lectins in a binding assay to GalNAc-glycopeptidessurprisingly showed similar inhibition with GalNAcα-benzyl andGalNAcβ-benzyl. This indicates that these lectins in contrast to manylectins including Helix Pomatia fail to distinguish the anomericconfiguration of the monosaccharide hapten recognized. βGalNAc is a rarelinkage in mammalian glycoproteins and is found only in N-linkedglycoproteins in man associated with the hormone specific glycosylationpattern where it generally is sulphated. Although, βGalNAc is found inboth ganglioseries (GalNAcβ1-4Galβ1-4Glcβ1-Cer) and globoseries(GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-Cer) it is expected that treatment ofcells with 2 mM GalNAcβ-benzyl will not interfere significantly withglycosylation. This is based on the findings that theβ1,3galactosyltransferases (β3Gal-T4 and β3Gal-T5, respectively)involved in extending βGalNAc in these two glycolipid structures show noor very poor activity with GalNAcβ-benzyl^(57,58). The only βGalNAccontaining structure in O-linked glycosylation is found in the bloodgroup related Sda structure(GalNAcβ1-4(Neuα2-3)Galβ1-3GalNAcα1-O-Ser/Thr) which has very restrictedexpression⁵⁹.

[0127] We therefore tested if GalNAcβ-benzyl treatment of cells showedthe same effects as GalNAcα-benzyl treatment. As shown in FIGS. 16-17(lane 5) GalNAcβ-benzyl treatment does not interfere withO-glycosylation in contrast to GalNAcα-benzyl (lane 4), as no differencein SDS-PAGE migration is observed. However, GalNAcα-benzyl, as well asGalNAcβ-benzyl treatment of cells, produces similar significantreduction in expression of MUC1 FIGS. 16-17. This shows surprisingly andfor the first time that GalNAcβ-benzyl represents a selective inhibitorof mucin transport and secretion. GalNAcβ-benzyl is a novel preferredinhibitor of transport, surface expression and secretion ofO-glycosylated proteins and mucins, because it does not interfere withthe O-glycosylation extension process. GalNAcβ-benzyl is not expected toaccumulate biosynthetic oligosaccharide products similar to those foundwith GalNAcα-benzyl treatment⁴⁶. The finding that GalNAcβ-benzyl exertsthese effects on mucin expression combined with the finding that itinhibits polypeptide GalNAc-transferases strongly indicate that themechanism by which GalNAcα- and GalNAcβ-benzyl inhibits mucin expressionis through inhibition of GalNAc-transferase lectins. This supports thesecond hypothesis articulated above. Polypeptide GalNAc-transferaselectins thus represent prime targets for intervention with mucinsecretion and cell surface expression, and GalNAcβ-benzyl represents anovel selective prototype inhibitor for such intervention.

[0128] Preferred compounds for inhibition of GalNAc-transferase lectinsare inactive as acceptor substrates for glycosyltransferases. Inparticular, the following glycosyltransferase activities: core 1UDP-Gal:GalNAc-peptide β1,3galactosyltransferases,CMP-NeuAc:GalNAc-peptide α2,6sialyltransferases, andUDP-GlcNAc:β1,3N-acetylglucosaminyltransferases involved inO-glycosylation, are inactive with the preferred inhibitory compounds.Examples of such inhibitory compounds are GalNAcα1-O-benzyl withsubstitution of hydroxyl groups at C3 and/or C6 with methyl or acetylgroups to block acceptor sites.

[0129] The methods described herein are designed to identify substancesand compounds that bind to and or modulate the biological activity of apolypeptide GalNAc-transferase or GalNAc-transferase lectin, includingsubstances that interfere with or enhance the activity of a polypeptideGalNAc-transferase lectin. GalNAc-transferase lectins may be used in theform of a truncated lectin domain as shown in Example 8, as a secretedGalNAc-transferase enzyme as shown in Example 8, or as a truncatedprotein or fusion protein with or without catalytic activity but withretained lectin domain and carbohydrate binding activity.

[0130] Agents that modulate a polypeptide GalNAc-transferase orGalNAc-transferase lectin can be identified based on their ability toassociate with such a transferase or lectin. Therefore, the inventionalso provides a method of identifying agents that associate with apolypeptide GalNAc-transferase or GalNAc-transferase lectin. Agentsidentified using the method of the invention may be isolated, cloned andsequenced using conventional techniques. An agent that associates with apolypeptide GalNAc-transferase or GalNAc-transferase lectin may be anagonist or antagonist of the biological or immunological activity of thetransferase or lectin.

[0131] Agents that can associate with a polypeptide GalNAc-transferaseor GalNAc-transferase lectin may be identified by reacting suchGalNAc-transferase or GalNAc-transferase lectin with a test substance,which potentially associates with a polypeptide GalNAc-transferase orlectin under conditions which permit the association, and removingand/or detecting the associated GalNAc-transferase or lectin andsubstance. The substance—GalNAc-transferase or substance-lectin complex,free substance, or non-complexed polypeptide may be assayed. Conditions,which permit the formation of substance—GalNAc-transferase orsubstance-lectin complexes, may be selected having regard to factorssuch as the nature and amounts of the substance and the polypeptide.

[0132] The substance-transferase or substance-lectin complex, freesubstance or non-complexed transferase or lectin may be isolated byconventional isolation techniques, for example, salting out,chromatography, electrophoresis, gel filtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, orcombinations thereof. To facilitate the assay of the components, alabelled antibody against the transferase or the substance or a labelledlectin or a labelled substance may be utilized. The antibodies, lectins,or test substances may be labelled with a detectable substance asdescribed above.

[0133] A polypeptide GalNAc-transferase or GalNAc-transferase lectin, ora test substance used in the method of the invention may beinsolubilized. For example, a lectin, transferase, or a test substancemay be bound to a suitable carrier such as agarose, cellulose, dextran,Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carriermay be in the shape of, for example, a tube, test plate, beads, disc,sphere etc. The insolubilized lectin, transferase or substance may beprepared by reacting the material with a suitable insoluble carrierusing known chemical or physical methods, for example, cyanogen bromidecoupling.

[0134] The invention also contemplates a method for evaluating an agentfor its ability to modulate the biological activity of a polypeptideGalNAc-transferase or GalNAc-transferase lectin by assaying for anagonist or antagonist (i.e. enhancer or inhibitor) of the association ofthe transferase or lectin with a substance that interacts with thepolypeptide (e.g. carbohydrate binding site or parts thereof). The basicmethod for evaluating whether an agent is an agonist or antagonist ofthe association of a polypeptide GalNAc-transferase or lectin and asubstance that associates with the transferase or lectin is to prepare areaction mixture containing the transferase lectin and the substanceunder conditions which permit the formation of substance-transferase orsubstance-lectin complexes, in the presence of a test agent. The testagent may be initially added to the mixture, or may be added subsequentto the addition of the transfearse or lectin and substance. Controlreaction mixtures without the test agent or with a placebo are alsoprepared. The formation of complexes is detected and the formation ofcomplexes in the control reaction, but not in the reaction mixture,indicates that the test agent interferes with the interaction of thetransferase/lectin and substance. The reactions may be carried out inthe liquid phase or the transferase/lectin, substance, or test agent maybe immobilized as described herein.

[0135] It will be understood that the agonists and antagonists, i.e.enhancers and inhibitors, that can be assayed using the methods of theinvention may act on one or more of the interaction sites on thetransferase or lectin or substance including agonist binding sites,competitive antagonist binding sites, non-competitive antagonist bindingsites or allosteric sites. It will also be understood that competitiveassays, in addition to direct assays, can be used to screen for andidentify the agents of the present invention.

[0136] The invention also makes it possible to screen for antagoniststhat inhibit the effects of an agonist of the interaction of apolypeptide GalNAc-transferase or GalNAc-transferase lectin with asubstance capable of associating with the lectin. Thus, the inventionmay be used to assay for an agent that competes for the same interactingsite of a polypeptide GalNAc-transferase lectin.

[0137] Test compounds are screened from, for example, large libraries ofsynthetic or natural compounds. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds. Examples of available libraries are synthetic compoundlibraries are commercially available from Maybridge Chemical Co.(Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), BrandonAssociates (Merrimack, N.H.), and Microsource (New Milford, Conn.).

[0138] Agents which are effective in modulating a polypeptideGalNAc-transferase lectin can be identified, based on their ability tointerfere with or enhance the lectin mediated binding capacity of theGalNAc-transferase protein or fragment hereof containing the lectinregion. Therefore, the invention provides a method for evaluating a testsubstance for its ability to modulate the binding capacity of apolypeptide GalNAc-transferase lectin comprising

[0139] (a) reacting a binding substrate with a GalNAc-transferase orlectin polypeptide or fragment hereof in the presence of a testsubstance;

[0140] (b) measuring the amount of binding substrate bound to theGalNAc-transferase polypeptide or fragment hereof, and

[0141] (c) carrying out steps (a) and (b) in the absence of the testsubstance to determine if the substance interferes with or enhances thebinding by the polypeptide GalNAc-transferase.

[0142] Suitable binding substrates for use in the methods of theinvention are polypeptides, glycopolypeptides, or glycoproteins, whichare either synthetic or naturally occurring structures. TheGalNAc-transferase lectin polypeptide may be obtained from naturalsources or produced using recombinant methods as described andreferenced herein.

[0143] The binding or modifying substrates or acceptor or donorsubstrates may be labelled with a detectable substance as describedherein, and the interaction of the polypeptide of the invention with thebinding or modifying substrates will give rise to a detectable change.The detectable change may be colorimetric, photometric, radiometric,potentiometric, etc. The GalNAc-transferase lectin polypeptide isreacted with the binding or modifying substrates at a pH and temperatureeffective for the polypeptide to bind the substrates, and wherepreferably one of the components is labeled, to produce a detectablechange. It is preferred to use a buffer with the substrates to maintainthe pH within the pH range effective for the polypeptides. The bufferand substrates may be used as an assay composition. Other compounds suchas EDTA and detergents may be added to the assay composition.

[0144] The reagents suitable for applying the methods of the inventionto evaluate agents that modulate a polypeptide GalNAc-transferase orGalNAc-transferase lectin may be packaged into convenient kits providingthe necessary materials packaged into suitable containers. The kits mayalso include suitable supports useful in performing the methods of theinvention.

[0145] Agents that modulate polypeptide GalNAc-transferase(s) orGalNAc-transferase lectin(s) can also be identified by treatingimmortalized cells which express the transferase(s) with a testsubstance, and comparing the intracellular transport, degradation,surface expression, or secretion of O-glycosylated proteins, mucins, andglycoproteins performed of the cells with those of the cells in theabsence of the test substance and/or with immortalized cells which donot express the transferase(s). Examples of immortalized cells that canbe used include human cell lines, Chinese hamster ovary (CHO) cells andmutant cells CHO ldlD⁵³, which express polypeptide GalNAc-transferase(s)or lectin(s) and produce cell membrane bound or secereted forms of thehuman mucin MUC1. In the absence of an inhibitor the cells will produceand transport MUC1 to the cell surface or secrete MUCI into the growthmedium. Substances that reduce the cell surface expression or thequantity of MUC1 in the medium may be considered an inhibitor.

[0146] The agents identified by the methods described herein, may beused for modulating the biological activity of a polypeptideGalNAc-transferase or a GalNAc-transferase lectin, and they may be usedas prototype drugs in the treatment of conditions mediated by apolypeptide GalNAc-transferase or GalNAc-transferase lectin and indesigning further substances effective to treat such conditions. Inparticular, they may be used to alter density of O-glycosylation onglycoproteins and mucins produced by cells, the intracellular transportand surface expression of glycoproteins and mucins, the secretion ofglycoproteins and mucins, and other functions governed by thepolypeptide GalNAc-transferases and their lectins in transport andsecretion of glycoproteins and mucins.

[0147] Therefore, the present invention has potential application in thetreatment of various disorders associated with aberrant O-glycosylationand/or mucin production in mammals, preferably humans. Such disordersinclude the following: tumors and cancers, lungs diseases associatedwith mucous accumulation such as asthma, chronic bronchitis, smoker'slung, cystic fibrosis, diseases of exocrine glands associated withincreased or decreased mucin secretion such as Sjøgrens syndrome, drymouth etc. Other disorders include dysregulation of selectin-mediatedleukocyte trafficking and would include but not be limited to disordersinvolving autoimmunity, arthritis leukaemia's, lymphomas,immunosuppression, sepsis, wound healing, acute and chronic in action,cell mediated immunity, and the like.

[0148] The agents identified by the methods described herein, havepotential application in treatment of tumors including inhibition oftumor metastasis and growth and/or regression of same. Tumor metastasismay be inhibited by inhibiting the adhesion of circulating cancer cells.The agents of the invention have particular potential application may beespecially useful in the treatment of various forms of neoplasia such asleukaemias, lymphomas, melanomas, adenomas, sarcomas, and carcinomas ofsolid tissues in patients. In particular the composition may be used fortreating malignant melanoma, pancreatic cancer, cervico-uterine cancer,cancer of the liver, kidney, stomach, lung, rectum, breast, bowel,gastric, thyroid, neck, cervix, salivary gland, bile duct, pelvis,mediastinum, urethra, bronchogenic, bladder, esophagus and colon, andKaposi's Sarcoma which is a form of cancer associated with HIV-infectedpatients with Acquired Immune Deficiency Syndrome (AIDS). The substancesetc. are particularly useful in the prevention and treatment of tumorsof lining mucosa and glands and the metastases derived from thesetumors.

[0149] Accordingly, the various agents may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. Bybiologically compatible form suitable for administration in vivo ismeant a form of the agent to be administered in which any toxic effectsare outweighed by the therapeutic effects. The agents may beadministered to living organisms including humans, and animals.Administration of a therapeutically active amount of the pharmaceuticalcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of an agent mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of pharmaceutical compositionor polypeptide to elicit a desired response in the individual. Dosageregima may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

[0150] The active agent may be administered in a convenient manner suchas by injection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active agent may be coated in amaterial to protect the agent from the action of enzymes, acids andother natural conditions that may inactivate it.

[0151] The compositions described herein can be prepared by methodsknown per se for the preparation of pharmaceutically acceptablecompositions which can be administered to subjects, such that aneffective quantity of the active agent is combined in a mixture with apharmaceutically acceptable vehicle. Suitable vehicles are described,for example, in Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA1985). On this basis, the compositions include, albeit not exclusively,solutions of the agents in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

[0152] The phrase “pharmaceutically acceptable” refers to molecularentities and compositions that are physiologically tolerable and do nottypically produce an allergic or similar untoward reaction (for example,gastric upset, dizziness and the like) when administered to anindividual. Preferably, and particularly where a immunogenic compositionis used in humans, the term “pharmaceutically acceptable” denotesapproved by a regulatory agency (for example, the U.S. Food and DrugAgency) or listed in a generally recognized pharmacopeia for use inanimals (for example, the U.S. Pharmacopeia).

[0153] Toxicity and therapeutic efficacy of compounds can be determinedby standard pharmaceutical procedures, for example in cell cultureassays or using experimental animals to determine the LD50 and the ED50.The parameters LD50 and ED50 are well known in the art, and refer to thedoses of a compound that are lethal to 50% of a population andtherapeutically effective in 50% of a population, respectively. The doseratio between toxic and therapeutic effects is referred to as thetherapeutic index and may be expressed as the ratio: LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used. However, in suchinstances it is particularly preferable to use delivery systems thatspecifically target such compounds to the site of affected tissue so asto minimize potential damage to other cells, tissues or organs and toreduce side effects.

[0154] Data obtained from cell culture assay or animal studies may beused to formulate a range of dosages for use in humans. The dosage ofcompounds used in therapeutic methods of the present inventionpreferably lie within a range of circulating concentrations thatincludes the ED50 concentration but with little or no toxicity (e.g.,below the LD50 concentration). The particular dosage used in anyapplication may vary within this range, depending upon factors such asthe particular dosage form employed, the route of administrationutilized, the conditions of the individual (e.g., patient), and soforth.

[0155] Non-human animals include, without limitation, laboratory animalssuch as mice, rats, rabbits, hamsters, guinea pigs, etc.; domesticanimals such as dogs and cats; farm animals such as sheep, goats, pigs,horses, and cows; and non-human primates.

[0156] A therapeutically effective dose may be initially estimated fromcell culture assays and formulated in animal models to achieve acirculating concentration range that includes the IC50. The IC50concentration of a compound is the concentration that achieves ahalf-maximal inhibition of symptoms (e.g., as determined from the cellculture assays). Appropriate dosages for use in a particular individual,for example in human patients, may then be more accurately determinedusing such information.

[0157] Measures of compounds in plasma may be routinely measured in anindividual such as a patient by techniques such as high performanceliquid chromatography (HPLC) or gas chromatography.

[0158] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients.

[0159] Thus, the compounds and their physiologically acceptable saltsand solvates may be formulated for administration by the routesdescribed above.

[0160] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0161] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0162] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0163] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0164] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0165] The compositions may, if desired, be presented in a pack ordispenser device that may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0166] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labelled for treatment of anindicated condition. For administration of an inhibitor of a polypeptideGalNAc-transferase, such labelling would include amount, frequency, andmethod of administration.

[0167] The use of inhibitors of the lectin domain mediated activities ofthe above mentioned polypeptide GalNAc-transferase isoforms and otherisoforms allows for unique selective inhibition of these functions invitro and in vivo in cells and organisms. This is desirable inmanipulating the density of O-glycans, e.g. changing high densityO-glycosylated tumour-associated MUC1 to low density normal MUC1 incells. Further this is desirable in inhibiting any adhesive role thelectin domains may play in Golgi transport and intracellular sorting.

[0168] Preferred agents for inhibition of GalNAc-transferase lectins areinactive as acceptor substrates for glycosyltransferases. In particular,the following glycosyltransferase activities are inactive with thepreferred inhibitory compounds: core 1 UDP-Gal:GalNAc-peptideβ1,3-galactosyltransferases, CMP-NeuAc:GalNAc-peptideα2,6-sialyltransferases, andUDP-GlcNAc:β1,3N-acetylglucosaminyltransferases involved inO-glycosylation. Examples of such inhibitory compounds areGalNAcα1-O-benzoyl with substitution of hydroxyl groups at C3 and/or C6by methyl or acetyl groups to block acceptor sites.

[0169] Agents which are effective in modulating a polypeptideGalNAc-transferase can be identified based on their ability to interferewith or enhance the activity of the transferase. Therefore, theinvention provides a method for evaluating a test substance for itsability to modulate the activity of a polypeptide GalNAc-transferasecomprising

[0170] (a) reacting an acceptor substrate and a donor substrate for aGalNAc-transferase polypeptide in the presence of a test substance;

[0171] (b) measuring the amount of donor substrate transferred toacceptor substrate, and

[0172] (c) carrying out steps (a) and (b) in the absence of the testsubstance to determine if the substance interferes with or enhancestransfer of the sugar donor to the acceptor by the polypeptideGalNAc-transferase.

[0173] Suitable acceptor substrates for use in the methods of theinvention are polypeptides, glycopolypeptides, or glycoproteins whichare either synthetic or naturally occurring structures. Acceptors willgenerally comprise the hydroxyamino acids serine and/or threonine. Thedonor substrate may be a nucleotide sugar, dolichol-phosphate-sugar ordolichol-pyrophosphate-oligosaccharide, for example, uridinediphospho-N-acetylgalactosamine (UDP-GalNAc), uridinediphospho-galactose (UDP-Gal), or derivatives or analogs thereof. TheGalNAc-transferase polypeptide may be obtained from natural sources orproduced using recombinant methods as described and referenced herein.

[0174] These and other embodiments of the present invention aredescribed in more detail below. The following examples are intended tofurther illustrate the invention without limiting its scope.

EXAMPLES Example 1

[0175] The MUC1 glycopeptide specificity of GalNAc-T4 is not directed bya specific glycoform.

[0176] The GalNAc-T4 isoform displays enzyme activity which, in additionto showing activity with some peptide substrates, exhibits uniqueactivity with glycopeptides where prior glycosylation is a prerequisitefor activity (8). GalNAc-T4 is unique in that it is the onlyGalNAc-transferase isoform identified so far that can complete theO-glycan attachment to all five acceptor sites in the 20 amino acidtandem repeat sequence (HGVTSAPDTRPAPGSTAPPA) of the breast cancerassociated mucin, MUC1. GalNAc-T4 transfers GalNAc to at least two sitesnot used by other GalNAc-transferase isoforms on the GalNAc₄TAP24glycopeptide (TAPPAHGVTSAPDTRPAPGSTAPP, GalNAc attachment sitesunderlined) (8). An activity such as that exhibited by GalNAc-T4 appearsto be required for production of the glycoform of MUC1 expressed bycancer cells where all potential sites are glycosylated (10). In orderto analyse activity of GalNAc-T4 with MUC1 derived GalNAc-peptides indetail different glycoforms of 24/25-mer peptides (TAP24/25) by usingdifferent GalNAc-transferase isoforms to catalyse glycosylation ofselected sites in combination with valine substitutions of acceptorsites were prepared. Surprisingly, analysis of the substrate specificityof GalNAc-T4 with different glycoforms of MUC1 revealed that GalNAc-T4did not show a requirement for any single site of GalNAc attachment(FIG. 1). By the contrary, there was only a requirement for at least oneof the three sites to be glycosylated. Thus, substitution of any one ofthe sites glycosylated in the GalNAc₄TAP24/25 glycopeptide by valine didnot affect activation of GalNAc-T4 activity for glycopeptides. Catalyticactivity with certain sites was affected by site specific modifications,in particular glycosylation of S in -VTSA- or -GSTA- was influenced byglycosylation at adjacent and distant sites. This result suggested thata unique and novel triggering event of GalNAc-T4 activity existed in thepresence of the glycosylated MUC1 substrate. This activity could not beascribed to simple conformational changes in the acceptor substrateinduced by the glycosylation. This surprising finding led us tohypothesise that a triggering event that was independent of the generalcatalytic activity of the enzyme led to acquisition of specificity forGalNAc-glycopeptides.

Example 2

[0177] The lectin domain of GalNAc-T4 selectively directs its MUCIglycopeptide specificity

[0178] One potential candidate for such a triggering event ofglycopeptide activity was the lectin domain, which was previously shownby mutational analysis to have no significant affect on the activity ofthe GalNAc-T1 isoform (3). Since GalNAc-T4 exhibits both glycosylationindependent and glycosylation dependent activities, it offers a modelsystem to analyse the different specificities as separate functions.Hagen et al. (3) originally demonstrated that critical substitutions inthe lectin domain of GalNAc-T1 have little affect on catalytic activity(reduction by 10-50%) with peptide substrates, while substitutions inthe catalytic domain destroyed activity (FIG. 2, Panel A). It waspredicted that mutation of an aspartate residue adjacent to a conservedCLD motif in the lectin domain to histidine (D444H in GalNAc-T1corresponding to D459H in GalNAc-T4) would destroy lectin function basedon analysis of ricin (29), but mutation of this residue (D444H) inGalNAc-T1 only appeared to reduce activity by approximately 50 %. Totest if the lectin domain influenced glycopeptide specificity ofGalNAc-T4, recombinant secreted forms of GalNAc-T4^(459D) and -T4^(459H)were prepared. GalNAc-T4^(459D) and -T4^(459H) exhibited essentially thesame specific activity with several unglycosylated peptides, inagreement with the results obtained for GalNAc-T1 (3) (illustrated for aPSGL-1 substrate in FIG. 2, Panel B). In contrast, the glycopeptidespecificity of mutant GalNAc-T4^(459H) was selectively affected by theintroduced mutations. Glycopeptides derived from tandem repeats of MUC1,MUC2 and MUC5AC (7) were virtually inactive as substrates, as isillustrated in FIG. 2 (Panel C), which depicts assays with aGalNAc₃TAP25V21 glycopeptide. Essentially identical results wereobserved with unsubstituted TAP24 and GalNAc₄TAP24 glycopeptide. Theseresults surprisingly demonstrates that the lectin domain is required forthe glycopeptide specificity of enzyme activity, but not for activitywith naked peptide substrates. This shows that the lectin domaintriggers the catalytic domain of GalNAc-T4 to act on GalNAc-glycopeptidesubstrates by an as yet unknown mechanism. Furthermore, it demonstratesthat the basic catalytic function and the triggering event areindependent properties associated with distinct domains ofGalNAc-transferases.

Example 3

[0179] The lectin domain of GalNAc-T4 functions as a lectin and hasselective specificity for GalNAc

[0180] In order to determine if actual carbohydrate binding contributedto the function of the lectin domain, we analysed whether triggering ofglycopeptide specificity could be blocked by specific carbohydrates insolution. We could not detect direct binding of GalNAc-T2 and -T4 tofree GalNAc using conventional binding assays presumably due to lowaffinity. More sensitive analyses will be required to demonstratebinding. However, as shown in FIG. 3 (Panel A) the glycosylationdependent specificity of GalNAc-T4 was almost completely inhibited byincubation with 0.23 M free GalNAc, whereas other sugars, Gal, GlcNAc,or Fuc, failed to show significant inhibition. Assays with 50 mM sugarsgave the same pattern, but with less (approximately 50%) inhibition byGalNAc (not shown). Furthermore, similar inhibition was found with 10 mMα-D-GalNAc-1-benzyl, whereas αGlcNAc-benzyl did not inhibit catalyticactivity. None of the sugars had significant affects on theglycosylation independent activities of GalNAc-T4^(459D) or -T4^(459H),when assayed with naked peptides (FIG. 3, Panel B). This demonstratesthat the lectin domain of GalNAc-T4 must bind to GalNAc and contributesto the ability of GalNAc T4 to catalyse glycosylation of glycopeptides.It further demonstrates examples of inhibitors that selectively blockthe GalNAc-peptide substrate specificity of GalNAc-T4. The finding thatneither Gal nor Galβ1-3GalNAcα1-benzyl produced significant inhibitioncompared to GalNAc suggests that the second step of O-glycosylation(extension of the oligosaccharide side chains), which is catalysed bythe β3galactosyltransferase forming the core 1 structureGalβ1-3GalNAcα1-O-Ser/Thr, may block the functional activity of thelectin domain of GalNAc-T4. Thus, once the O-glycan processing stepinvolving elongation to the core 1 structure is accomplished, GalNAc-T4would not be capable of catalysing glycosylation of glycopeptides. Thissuggests that O-glycan elongation/branching and O-glycan density may beregulated by competition among GalNAc-transferases (lectin domain) andthe glycosyltransferases involved in O-glycan extension, especially thecore 1 synthase β3Gal-transferase.

Example 4

[0181] The lectin domain of GalNAc-T7 functions as a lectin and hasselective specificity for GalNAc and Galβ1-3GalNAc

[0182] GalNAc-T7 exhibits exclusive glycopeptide specificity and nounsubstituted acceptor peptide substrates have been identified thus far(7). GalNAc-T7 has a different glycopeptide substrate specificity thanGalNAc-T4 and does not function with MUC1 derived glycopeptides. Thebest substrate identified to date is derived from the tandem repeatregion of rat submaxillary gland mucin (30). The activity of GalNAc-T7with GalNAc2-3EA2 was significantly inhibited by benzyl-αGalNAc,benzyl-βGal, and the Galβ1-3GalNAcα1-benzyl disaccharide core 1structure at 5 mM concentrations (Table I). TABLE I Inhibition ofGalNAc-T7 activity with GalNAc2-3EA2 substrate Activity (nmol/min/ml) inthe presence of inhibitors (5 mM) None bz-αMan bz-βGal bz-αGalNAcGalβ-3GalNAcα1-bz 6.8 6.7 4.7 5.4 4.5

Example 5

[0183] The lectin domain of GalNAc-T2 is functional and has selectivespecificity for GalNAc and the MUC2 and MUC5AC tandem repeat peptides

[0184] GalNAc-T2 exhibits galactosyltransferase activity in the presenceof the Muc2 acceptor substrate (28). Furthermore, testing a panel ofpeptide substrates it was found that GalNAc-T2 also utilized Muc7 and tolesser degree the EA2 peptide in the presence of UDP-Gal (GalNAc-T2activity with UDP-Gal: Muc2, 90 nmol/min/ml; Muc7, 13 nmol/min/ml; EA2,1.5 nmol/min/ml). The galactosyltransferase activity with Muc2 substratewas selectively inhibited by GalNAc and not other sugars. TABLE IIInhibition of GalNAc-T2 activities with Muc2 acceptor substrate Activity(nmol/min/ml) in the presence Donor of inhibitors (230 mM) SubstrateNone GalNAc GlcNAc Gal Fuc UDP-GalNAc 340 300 310 300 370 UDP-Gal 90 2468 89 89

[0185] Since the galactosyltransferase activity exhibited by GalNAc-T2exhibits an entirely different acceptor substrate pattern than theN-acetylgalactosaminyltransferase activity, it is concluded that thelectin domain exhibits peptide binding specificity in addition toGalNAc. Hence, the mechanism of activation resemble that of theglycopeptide specificity of GalNAc-T4 only the trigger is a peptidesequence motif comprised in the Muc2 and Muc7 peptide sequences.Inhibitors to the lectin domain of GalNAc-T2 will block its ability tobind nascent unglycosylated MUC2 mucin polypeptides and hence affect apotential chaperone effect of GalNAc-T2 in Golgi.

Example 6

[0186] The lectin domain of GalNAc-T3 is functional and has selectivespecificity for GalNAc and the MUC5AC and rat submaxillary tandem repeatpeptides

[0187] GalNAc-T3 was found also to exhibit galactosyltransferaseactivity but only in the presence of the EA2 acceptor substrate(GalNAc-T2 activity with UDP-Gal: Muc2, 0 nmol/min/ml; Muc7, 0.1nmol/min/ml; EA2, 6.8 nmol/min/ml). The galactosyltransferase activitywith Muc2 substrate was selectively inhibited by GalNAc and not othersugars. TABLE III Inhibition of GalNAc-T3 activities with EA2 acceptorsubstrate Activity (nmol/min/ml) in the Donor presence of inhibitors(230 mM) Substrate None GalNAc GlcNAc Gal Fuc UDP-GalNAc 34 35 32 33 34UDP-Gal 6.3 2.5 7 7.3 6.5

[0188] The lectin domain of GalNAc-T3 resemble that of GalNAc-T2 inbinding to peptide sequences although the sequence motif must bedifferent and partly contained in the EA2 sequence.

[0189] Materials and Methods.

[0190] The following subsections describe the materials and methods usedin Examples 1-6.

[0191] Enzyme Reaction Conditions and Substrates

[0192] Standard reaction mixtures (50 μl final volume) contained 25 mMcocadylate (pH 7.4), 10 mM MnCl₂, 0.25% Triton X-100, 200 μMUDP-[¹⁴C]-GalNAc (2,000 cpm/nmol) (Amersham), 200-500 μM acceptorpeptides. Products were quantified by scintillation counting afterchromatography on Dowex-1, octadecyl silica cartridges (Bakerbond), orHPLC (PC3.2/3 or mRPC C2/C18 SC2.1/10 Pharmacia, Smart System). Acceptorpeptides included five variants of TAP25(TAPPAHGV(T/V)SAPDTRPAPG(S/V)(T/V)APPA) and TAP24(TAPPAHGVTSAPDTRPAPGSTAPP) derived from the human MUC1 tandem repeat(31); MUC2 (PTTTPISTTTMVTPTPTPTC) derived from human intestinal mucinMUC2 (32); MUC5AC (Ac-SAPTTSTTSAPT) derived from human respiratory glandmucin MUC5AC (33); MUC7 (Ac-CPPTPSATTPAPPSSSAPPETTAA) derived from humansalivary gland mucin MUC7 (34); EA2 (PTTDSTTPAPTTK) derived from ratsubmandibular gland mucin (30); VTHPGY (Ac-PFVTHPGY) derived from humanfibronectin (35); Zonadhesin (PTERTTTPTKRTTTPTIR) derived from humanzonadhesin (36); OSM fragment (LSESTTQLPGGGPGCA) derived from ovinesubmaxillary mucin (37); hCG-β (PRFQDSSSSKAPPPLPSPSRLPG) derived fromhuman chorionic gonadotropin β-subunit (38); MUC1b (RPAPGSTAPPA) derivedfrom MUC1 and PSGL-1b (Ac-QATEYEYLDYDFLPETEPPEM) derived from theN-terminus of P-selectin ligand-1 (39). GalNAc-glycopeptides of MUC2,MUC5AC and MUC7 were produced using cold UDP-GalNAc and purified humanrecombinant GalNAc-T1 and -T2 (28). Different GalNAc-glycoforms of EA2were produced by limiting the ratio of UDP-GalNAc to 2 moles, 3 moles, 4moles or 5 moles per mole of acceptor peptide. Glycopeptides werepurified on Supelclean LC-18 columns (1 ml, Supelco), and the number ofGalNAc residues incorporated evaluated by MALDI-TOF mass spectrometry.The enzyme sources used were semipurified as previously described bysuccessive sequential ion-exchange chromatographies on Amberlite (IRA95,Sigma) or DEAE Sephacel (Pharmacia), S-Sepharose Fast Flow (Pharmacia),and Mini-S™ (PC 3.2/3, Pharmacia) using the Smart System (Pharmacia)(28). Secreted GalNAc-T4 was obtained from a stably transfected CHO line(CHO/GalNA-T4/21A) (8) grown in roller bottles in HAMS F12 supplementedwith 10% Fetal Bovine Serum. The experiments illustrated in FIG. 1 wasperformed with recombinant secreted GalNAc-T4 obtained from a stablytransfected CHO line (8). Experiments illustrated in FIGS. 2 and 3 wereperformed with secreted GalNAc-T4 expressed in High Five cells grown inserum-free medium (8). Structural analysis of glycopeptides wereperformed by a combination of PFPA (pentafluoropropionic acid)hydrolysis and MALDI-TOF mass spectrometry as previously described (40).Secreted GalNAc-T7 was obtained from infected High Five™ cells grown inserum-free medium (Invitrogen) in upright roller bottles shaken 140 rpmin waterbaths at 27° C. GalNAc-T7 was not purified by Mini-S as theyields from cationic chromatography were low due to its low pI (6.4).

[0193] Reaction Kinetics Monitored by Capillary Electrophoresis.

[0194] Reaction mixtures were modified to include 1.7 mM coldUDP-GalNAc, 25 μg acceptor peptides, and purified GalNAc-transferases ina final volume of 100 μl. The amount of GalNAc-transferase added wasadjusted so that the reaction with the appropriate peptide was nearcompletion in six hours. Reactions were incubated in the sample carouselof an Applied Biosystem model HT270 at 30° C. as described previously(28). Electrophoretograms were produced every 60 min, and after sixhours the reaction mixtures were separated by reverse phase HPLC forstructural determination. HPLC was performed on a Brawnlee ODS column(2.1 mm×30 mm, 5 μm particle size) (Applied Biosystems, Inc.) using alinear gradient (0-30%, 0.1 % TFA/ 0.08% TFA, 90% acetonitrile, 30 min)delivered by an ABI 130A micro-bore HPLC system (Perkin Elmer Inc).

[0195] Structural Analysis of Reaction Products

[0196] Glycopeptides were purified by HPLC and analysed by a combinationof PFPA (pentafluoropropionic acid, Sigma) hydrolysis and MALDI-TOF massspectrometry. Glycopeptides (50 pmol) were lyophilized in 500 μlEppendorf vials and placed in a 22 ml glass vial with a mininert valve(Pierce, Rockford, Ill.). A solution of 100 μl 20% PFPA (aqueous)containing 500 μg DTT was added to the bottom of the glass vial, whichwas then flushed with argon. The vial was evacuated to 1 mbar, andplaced in an oven at 90° C. for 60 min. The hydrolyzed samples werecentrifuged in a vacuum centrifuge for 15 min to remove remaining tracesof acid. Lyophilized samples were reconstituted in 0.1 % TFA to aconcentration of 1 pmol/μl. Mass spectra were acquired on eitherVoyager-DE or Voyager-Elite mass spectrometers equipped with delayedextraction (Perseptive Biosystem Inc.). The matrix used was2,5-dihydroxybenzoic acid (10 mg/ml, Hewlett-Packard) dissolved in a 2:1mixture of 0.1 % trifluoroacetic acid in 30 % aqueous acetonitrile(Rathburn Ltd.). Samples dissolved in 0. 1 % trifluoroacetic acid to aconcentration of approximately 80 fmol to 1 pmol/ml were prepared foranalysis by placing 1 μl of sample solution on a probe tip followed by 1μl of matrix. The hydrolyzed samples were prepared for MALDI analysisusing nano-scale reversed-phase columns (Poros R3, PerSeptiveBiosystem), according to previously described procedure (41). Sampleswere prepared by mixing 0.8 μl of total fraction volume 2 pmol ofhydrolyzed glycopeptides and 0.4 μl of matrix solution. Mass spectrawere acquired in reflector mode on a Voyager-Elite BiospectrometryWorkstation (PerSeptive Biosystems Inc., Framingham, Mass., USA)equipped with delayed ion extraction technology. Data processing wasperformed using software packages Perseptive-Grams (Galactic IndustriesCorp.) and protein analysis software GPMAW (htpp://www.welcome.to/gpmaw;Lighthouse data, Odense, Denmark)

[0197] Reaction Kinetics Monitored by Mass Spectrometry

[0198] MALDI-TOF time-course in terminal reactions were performed inreactions of 25 μl containing 2.5 nmol acceptor (glyco)peptide, 40 nmolUDP-GalNAc, and 0.4 μg GalNAc-T4. Sampling of reactions (1 μl) werepurified by nano-scale reversed-phase chromatography (Poros R3,PerSeptive Biosystem) and applied directly to the probe with matrix(41). The amount of GalNAc-transferase added was adjusted so that thereaction with the appropriate peptide was near completion in six hours.Reactions were incubated at 37° C. in a shaker bath. At times 0, 2hours, and 16 hours a 1 μl aliquot was taken and purified. Mass spectrawere acquired on either Voyager-DE mass spectrometer equipped withdelayed extraction (Perseptive Biosystem Inc.). The matrix used was2,5-dihydroxybenzoic acid (10 mg/ml, Hewlett-Packard) dissolved in a 2:1mixture of 0.1% trifluoroacetic acid in 30% aqueous acetonitrile(Rathburn Ltd.).

[0199] Construction, expression, purification, and analysis of a lectindomain mutant of GalNAc T4

[0200] The mutant GalNAc-T4^(459H) was prepared by multiplex PCR usingthe GalNAc-T4-sol construct that encodes residues 32-578 inserted intopT7T3U19 (8). Primers EBHC332 (5′-GTAGAGGGATCTCGTCTGAATGTTTACATTATA-3′(mutation underlined in bold)) and T7 (5′-TAATACGACTCACTATAGGG-3) wereused in a standard reaction under the following cycling conditions; 95°C. 45 s, 51° C. 5 s, 72° C. 1 min. For 18 cycles using a Tc 2400thermocycler (PE Biosystems, USA). The PCR product was digested withBstYI gel purified. 5 ng hereof was mixed with 10 ngpAcGP67-GalNAc-T4sol (8), and the mixture used to prime a “shuffle PCR”reaction using primers T7/EBHC201 (5′AAGCGGGCACCATATGCTCG-3′), usingstandard conditions and the following cycling conditions 95° C. 45 s,51° C. 5 s, 72° C. 1 min (5 cycles without primers, after which primerswere added and the reactions was cycled for an additional 17 cycles).The generated PCR product was digested with HindIII and inserted intoHindIII digested GalNAc-T4 pT7T3U19 construct described above. Themutated T4- construct was fully sequenced and the BamHI insert subclonedinto pAcGP67.

[0201] Wild-type and mutant constructs expressed in insect cells weresecreted in comparable yields, and the purified proteins migrated bySDS-PAGE identically. Quantification of purified proteins was done byCoomassie stained SDS-PAGE and titration of immunoreactivity with themonoclonal antibody, UH6(4G2) (8). GalNAc-T4^(459D) and -T4^(459H) werepurified to 0.04 μg/μl and 0.1 μg/μl with specific activities of 0.197U/mg and 0.24 U/mg with a MUC7 tandem repeat derived peptide (7),respectively. Wild-type GalNAc-T4 and mutant GalNAc-T4,GalNAc-T4^(459H), were analysed with unglycosylated peptides(represented by PSGL-1) or GalNAc glycosylated glycopeptides(represented by GalNAc3TAP25V21) in reactions of 25 μl containing 2.5nmol acceptor (glyco)peptide, 40 nmol UDP-GalNAc, and 0.4 μg GalNAc-T4.Time-course assays were motioned by MALDI-TOF. Sampling of reactions (1μl) were purified by nano-scale reversed-phase chromatography (Poros R3,PerSeptive Biosystem) and applied directly to the probe with matrix.Evaluation of inhibition of the glycopeptide specificity of wild-typeGalNAc-T4 with free sugars was performed to establish if the lectindomain recognized carbohydrate. Analysis was performed as above with0.23 M free GalNAc, Gal, GlcNAc, or Fuc, and the reaction was monitoredby MALDI-TOF. Further, analysis was performed with 10 mMα-D-GalNAc-1-benzyl, αGlcNAc-benzyl, and fully occupiedGalNAc-glycopeptide, GalNAc6TAP25, at 5 mM.

[0202] Inhibition of the GalNAc-Glycopeptide Activity of GalNAc-T7

[0203] GalNAc-T7 activity was analysed with GalNAc glycosylatedglycopeptides (represented by GalNAc₂₋₃EA2 (7)) in reactions of 25 μlcontaining 2.5 nmol acceptor (glyco)peptide, 40 nmol UDP-GalNAc, andpurified GalNAc-T7. Assays were performed with 0.23 M free GalNAc, Gal,GlcNAc, or Fuc, and the reaction product quantified by Dowex-1chromatography and scintillation counting. Further, analysis wasperformed with 10 mM α-D-GalNAc-1-benzyl, αGlcNAc-benzyl, and fullyoccupied GalNAc-glycopeptide, GalNAc6TAP25, at 5 mM.

[0204] Inhibition of lectin domains of GalNAc-transferases, GalNAc-T2and -T3, that do not exhibit glycopeptide specificities

[0205] GalNAc-T2 exhibits activity with UDP-Gal in the presence of theacceptor substrate Muc2 (28). Galactosyl transferring activities ofGalNAc-T1, -T2, and -T3, were assayed with a panel of acceptor peptidesin standard reaction mixtures containing 100 μM UDP-Gal instead ofUDP-GalNAc. GalNAc-T2 showed activity with Muc2 as well as low activitywith Muc7 and very low activity with EA2 acceptor substrates. GalNAc-T3showed activity with EA2 and lower activity with Muc7, but no activitywith other peptides tested. Since the activities with UDP-Gal do notcorrelate with the general acceptor substrate specificities of theseGalNAc-transferase isoforms found with the UDP-GalNAc donor substrate,it was tested if the lectin domains were involved. This was done byanalysing if free sugars could selectively inhibit the activities withUDP-Gal and not UDP-GalNAc. Assays were performed with 0.23 M freeGalNAc, Gal, GlcNAc, or Fuc, and the reaction product quantified byDowex-1 chromatography and scintillation counting. Further, analysis wasperformed with 10 mM α-D-GalNAc-1-benzyl, αGlcNAc-benzyl, and fullyoccupied GalNAc-glycopeptide, GalNAC6TAP25, at 5 mM.

Example 7. Cloning, Expression, and Purification of SolubleGalNAc-Transferase Proteins and Soluble GalNAc-Transferase Lectins.

[0206] Polypeptide GalNAc-transferases are highly conserved throughoutevolution. Orthologous relationships can be defined from man toDrosophila,⁶¹ and ortholgous members of all human polypetideGalNAc-transferase isoforms are clearly identifiable in mouse and rats,and likely all mammals.

[0207] Polypeptide GalNAc-transferases are predicted to be type IItransmembrane Golgi-resident proteins with a domain structure depictedin FIG. 2A. The N-terminal cytoplasmic tail, the hydrophic transmembranesignal sequence, and the stem region may be involved in directingGolgi-localization⁶⁰. The catalytic unit of the enzymes is approximately300-350 amino acid residues and highly conserved in primary sequenceamong isoforms and also throughout evolution of the gene family^(43,61).The C-terminal region of approximately 130 amino acids exhibitssimilarity with the galactose binding lectin, ricin. This region showlittle sequence similarity among isoforms and is poorly conserved inevolution⁴³.

[0208] Soluble, secreted expression constructs of humanGalNAc-transferases GalNAc-T1, -T2, -T3, -T4, -T6, -T7, and -T11 forbaculo-virus mediated expression in insect cells have been described indetail previously 7, 8, 26, 27, 43, 48. His-tagged soluble expressionconstructs for all human ppGalNAc-transferases, including novel genesdesignated GalNAc-T12, -T13, -T14, -T15, and -T16, were prepared usingPCR primers as listed in Table IV below. TABLE IV Primers used for PCRof soluble secreted GaINAc-transferase expression constructs. GaINAc-T1:EBHC121H: 5′-GCGGGATCCAGGACTTCCTGCTGCAGATC-3′ EBHC107B:5′-GCGGATCCTCAGAATATTTCTGGAAGGG-3′ GaINAc-T2: EBHC75D:5′-GCGGAATTCTTAAAAAGAAAGACCTTCATCACAGC-3′ EBHC68:5′-GCGGAATTCCTACTGCTGCAGGTTGAGC-3′ GaINAc-T3: EBHC219H:5′-GCGGGATCCAACGATGGAAAGGAACATG-3′ EBHC215:5′-AGCGGATCCAGGAACACTTAATCATTTTGGC-3′ GaINAc-T4: EBHC318:5′-GCGGGATCCTTTTCATGCCTCCGCAGGAGCC-3′ EBHC307:5′-GCGCGATCCGACGAAAGTGCTGTTGTGCTC-3′ GaINAc-T5: EBHC909:5′-GCGGGATCCTGCTTTAACTGGAGGGCTAGAGC-3′ EBHC907:5′-GCGGGATCCATCAGTTACACTTCAGGCTTC-3′ GaINAc-T6: EBHC514H:5′-GCGGGATCCCCTGGACCTCATGCTGGAGGCCATG-3′ EBHC511N:5′-AGCGGATCCTGGGGATGATCTGGGTCCTAGAC-3′ GaINAc-T7: EBHC1122H:5′-GCGAACCTTCAGGATGAGGGAAGACAGAGATG EBHC1116H:5′-GCGAAGCTTCTCTCTAAACACTATGGATCTTATTC-3′ GaINAc-T8: EBHC1820:5′-GCGGGATCCTCTCAAAGAAAGTATGAAATTAGC-3′ EBHC1821:5′-GCGGGATCCTCACTGGCTGTTGGTCTGACC GaINAc-T9: EBHC1320:5′-GCGGGATCCCTGCCGCCTGCAGGGCCGCTCCCAG-3′ EBHC1321:5′-GCGGGATCCTCAGTGCCGTCGGTGTTTCATCC-3′ GaINAc-T10: EBHC2520:5′-GCGGGATCCCCGCGAGCGGCAGCCCGACGGC-3′ EBHC2521:5′-GCGGGATCCTCAGTTCCTATTCAATTTTTC-3′ GaINAc-T11: EBHC629:5′-GCGAATTCGTGAAGTGACTCAGCCACTTAAG-3′ EBHC614:5′-GCGAATTCGTCTCTGTCAGACACGTGTC-3′ GaINAc-T12: EBHC1051:5′-GCGGGATCCGGCTCGGTGCTGCGGGCGCAGCG-3′ EBHC1032:5′-GCGGGATCCTCATAACATGCGCTCTTTGAAGAACC-3′ GaINAc-T13: EBHC2000:5′-GCGGGATCCGATGTTGCACVVTCCCCACCACACC-3′ EBHC2002:5′-GCGGGATCCTCATCGTTCATCCACAGCATTC-3′ GaINAc-T14: EBHC1720:5′-GCGGGATCCTCTGCTGCCTGCATTGAGGGCTG-3′ EBH21721:5′-GCGGGATCCTCATGTGCCCAAGGTCATGTTCC-3′ GaINAc-T15: EBHC412:5′-GCGGGATCCCAAGAGGAAGTTGGAGGTGCCG-3′ EBHC438:5′-GCGGGATCCCACCCCTCCTCAAGAGCTCACC-3′ GaINAc-T16: EBHC1913:5′-GCGGGATCCCTACTACTTATGGCACGACAACCC-3′ EBHC1912:5′-GCGTCATGTGTGTGGCAACAGCTGCCACTG-3′

[0209] Expression constructs were amplified by PCR using 20 ng plasmidDNA as template. Expand High Fidelity-kit (Roche) was used asrecommended by the manufacturer using an ABI2700 thermocycler (AppliedBiosystems). Products were digested with EcoRi (GalNAc-T2, -T11, -T12and -Ti6), BamHI (GalNAc-T1, -T3, -T4, -T5, -T6, -T8, -T9, -T10, -T13,-T14 and -T15) and HindIII (GalNAc-T7), and sub-cloned into the EcoRI orHindIII site of pBKS-HistagI or the BamHI site of pBKS-HistagII.PBKS-Histag-I and -II vectors were generated from pBluescrip(Stratagene), by inserting a fragment encoding 6×His, a thrombincleavage site, and a T7 antibody site. pBKS-Histag-I was modified withthe sequence5′-GCGGCCGCTCTAGAACTAGTGGATCCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGAATTCCGATATCAAGCTTATCGATACCGTCGACCTCGAG -3′.

[0210] pBKS-Histag-II was modified with the sequence:5′-GAATTCGCGGCCGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGATCCACTAGTTCTAGAGCGGCCG C -3′.

[0211] All construct were fully sequenced. His-tagged GalNAc-transferasepBKS-HIS-tag-I constructs were excised with NotI and XhoI (blunt-ended)or as for GalNAc-T11, with NotI and HindIII (blunt-ended) and sub-clonedinto the NotI/BgIII (blunt-ended) site of the pAcGP67A Baculo expressionvector (Pharmingen). His-tagged GalNAc-transferase pBKS-HIS-tag-IIconstructs were excised with NotI and inserted into the NotI site ofpAcGP67A Baculo expression vector.

[0212] The coding region for human polypeptide GalNAc-T12 has beensubmitted to GenBank/EBI Data Bank and assigned accession numberAJ132365: Human GalNAc-T12 DNA sequence:ATGTGGGGGCGCACGGCGCGGCGGCGCTGCCCGCGGGAACTGCGGCGCGGCCGGGAGGCGCTGTTGGTGCTCCTGGCGCTACTGGCGTTGGCCGGGCTGGGCTCGGTGCTGCGGGCGCAGCGTGGGGCCGGGGCCGGGGCTGCCGAGCCGGGACCCCCGCGCACCCCGCGCCCCGGGCGGCGCGAGCCGGTCATGCCGCGGCCGCCGGTGCCGGCGAACGCGCTGGGCGCGCGGGGCGAGGCGGTGCGGCTGCAGCTGCAGGGCGAGGAGCTGCGGCTGCAGGAGGAGAGCGTGCGGCTGCACCAGATTAACATCTACCTCAGCGACCGCATCTCACTGCACCGCCGCCTGCCCGAGCGCTGGAACCCGCTGTGCAAAGAGAAGAAATATGATTATGATAATTTGCCCAGGACATCTGTTATCATAGCATTTTATAATGAAGCCTGGTCAACTCTCCTTCGGACAGTTTACAGTGTCCTTGAGACATCCCCGGATATCCTGCTAGAAGAAGTGATCCTTGTAGATGACTACAGTGATAGAGAGCACCTGAAGGAGCGCTTGGCCAATGAGCTTTCGGGACTGCCCAAGGTGCGCCTGATCCGCGCCAACAAGAGAGAGGGCCTGGTGCGAGCCCGGCTGCTGGGGGCGTCTGCGGCGAGGGGCGATGTTCTGACCTTCCTGGACTGTCACTGTGAATGCCACGAAGGGTGGCTGGAGCCGCTGCTGCAGAGGATCCATGAAGAGGAGTCGGCAGTGGTGTGCCCGGTGATTGATGTGATCGACTGGAACACCTTCGAATACCTGGGGAACTCCGGGGAGCCCCAGATCGGCGGTTTCGACTGGAGGCTGGTGTTCACGTGGCACACAGTTCCTGAGAGGGAGAGGATACGGATGCAATCCCCCGTCGATGTCATCAGGTCTCCAACAATGGCTGGTGGGCTGTTTGCTGTGAGTAAGAAATATTTTGAATATCTGGGGTCTTATGATACAGGAATGGAAGTTTGGGGAGGAGAAAACCTCGAATTTTCCTTTAGGATCTGGCAGTGTGGTGGGGTTCTGGAAACACACCCATGTTCCCATGTTGGCCATGTTTTCCCCAAGCAAGCTCCCTACTCCCGCAACAAGGCTCTGGCCAACAGTGTTCGTGCAGCTGAAGTATGGATGGATGAATTTAAAGAGCTCTACTACCATCGCAACCCCCGTGCCCGCTTGGAACCTTTTGGGGATGTGACAGAGAGGAAGCAGCTCCGGGACAAGCTCCAGTGTAAAGACTTCAAGTGGTTCTTGGAGACTGTGTATCCAGAACTGCATGTGCCTGAGGACAGGCCTGGCTTCTTCGGGATGCTCCAGAACAAAGGACTAACAGACTACTGCTTTGACTATAACCCTCCCGATGAAAACCAGATTGTGGGACACCAGGTCATTCTGTACCTCTGTCATGGGATGGGCCAGAATCAGTTTTTCGAGTACACGTCCCAGAAAGAAATACGCTATAACACCCACCAGCCTGAGGGCTGCATTGCTGTGGAAGCAGGAATGGATACCCTTATCATGCATCTCTGCGAAGAAACTGCCCCAGAGAATCAGAAGTTCATCTTGCAGGAGGATGGATCTTTATTTCACGAACAGTCCAAGAAATGTGTCCAGGCTGCGAGGAAGGAGTCGAGTGACAGTTTCGTTCCACTCTTACGAGACTGCACCAACTCGGATCATCAGAAATGGTTCTTCAAAGAGCGCATGTTATGA

[0213] Human GalNAc-T12 amino acid sequence:MWGRTARRRCPRELRRGREALLVLLALLALAGLGSVLRAQRGAGAGAAEPGPPRTPRPGRREPVMPRPPVPANALGARGEAVRLQLQGEELRLQEESVRLHQINIYLSDRISLHRRLPERWNPLCKEKKYDYDNLPRTSVIIAFYNEAWSTLLRTVYSVLETSPDILLEEVILVDDYSDREHLKERLANELSGLPKVRLIRANKKKGLVRARLLGASAARGDVLTFLDCHCECHEGWLEPLLQRIHEEESAVVCPVIDVIDWNTFEYLGNSGEPQIGGFDWRLVFTWHTVPERERIRMQSPVDVIRSPTMAGGLFAVSKKYFEYLGSYDTGMEVWGGENLEFSFRIWQCGGVLETHPCSHVGHFSPSKLPTPRNKALANSVRAAEVWMDEFKELYYHRNPRARLEPFGDVTERKQLRDKLQCKDFKWFLETVYPELHVPEDRPGFFGMLQNKGLTDYCFDYNPPDENQIVGHQVILYLCHGMGQNQFFEYTSQKEIRYNTHQPEGCIAVEAGMDTLIMHLCEETAPENQKFILQEDGSLFHEQSKKCVQAARKESSDSFVPLLRDCTNSDHQKWFFKERML

[0214] The coding region for human polypeptide GalNAc-T13 has beensubmitted to GenBank/EBI Data Bank and assigned accession numberAR153422.

[0215] Human GalNAc-T13 DNA sequence:ATGCTCCTAAGGAAGCGATACAGGCACAGACCATGCAGACTCCAGTTCCTCCTGCTGCTCCTGATGCTGGGATGCGTCCTGATGATGGTGGCGATGTTGCACCCTCCCCACCACACCCTGCACCAGACTGTCACAGCCCAAGCCAGCAAGCACAGCCCTGAAGCCAGGTACCGCCTGGACTTTGGGGAATCCCAGGATTGGGTACTGGAAGCTGAGGATGAGGGTGAAGAGTACAGCCCTCTGGAGGGCCTGCCACCCTTTATCTCACTGCGGGAGGATCAGCTGCTGGTGGCCGTGGCCTTACCCCAGGCCAGAAGGAACCAGAGCCAGGGCAGGAGAGGTGGGAGCTACCGCCTCATCAAGCAGCCAAGGAGGCAGGATAAGGAAGCCCCAAAGAGGGACTGGGGGGCTGATGAGGACGGGGAGGTGTCTGAAGAAGAGGAGTTGACCCCGTTCAGCCTGGACCCACGTGGCCTCCAGGAGGCACTCAGTGCCCGCATCCCCCTCCAGAGGGCTCTGCCCGAGGTGCGGCACCCACTGTGTCTGCAGCAGCACCCTCAGGACAGCCTGCCCACAGCCAGCGTCATCCTCTGTTTCCATGATGAGGCCTGGTCCACTCTCCTGCGGACTGTACACAGCATCCTCGACACAGTGCCCAGGGCCTTCCTGAAGGAGATCATCCTCGTGGACGACCTCAGCCAGCAAGGACAACTCAAGTCTGCTCTCAGCGAATATGTGGCCAGGCTGGAGGGGGTGAAGTTACTCAGGAGCAACAAGAGGCTGGGTGCCATCAGGGCCCGGATGCTGGGGGCCACCAGAGCCACCGGGGATGTGCTCGTCTTCATGGATGCCCACTGCGAGTGCCACCCAGGCTGGCTGGAGCCCCTCCTCAGCAGAATAGCTGGTGACAGGAGCCGAGTGGTATCTCCGGTGATAGATGTGATTGACTGGAAGACTTTCCAGTATTACCCCTCAAAGGACCTGCAGCGTGGGGTGTTGGACTGGAAGCTGGATTTCCACTGGGAACCTTTGCCAGAGCATGTGAGGAAGGCCCTCCAGTCCCCCATAAGCCCCATCAGGAGCCCTGTGGTGCCCGGAGAGGTGGTGGCCATGGACAGACATTACTTCCAAAACACTGGAGCGTATGACTCTCTTATGTCGCTGCGAGGTGGTGAAAACCTCGAACTGTCTTTCAAGGCCTGGCTCTGTGGTGGCTCTGTTGAAATCCTTCCCTGCTCTCGGGTAGGACACATCTACCAAAATCAGGATTCCCATTCCCCCCTCGACCAGGAGGCCACCCTGAGGAACAGGGTTCGCATTGCTGAGACCTGGCTGGGGTCATTCAAAGAAACCTTCTACAAGCATAGCCCAGAGGCCTTCTCCTTGAGCAAGGCTGAGAAGCCAGACTGCATGGAACGCTTGCAGCTGCAAAGGAGACTGGGTTGTCGGACATTCCACTGGTTTCTGGCTAATGTCTACCCTGAGCTGTACCCATCTGAACCCAGGCCCAGTTTCTCTGGAAAGCTCCACAACACTGGACTTGGGCTCTGTGCAGACTGCCAGGCAGAAGGGGACATCCTGGGCTGTCCCATGGTGTTGGCTCCTTGCAGTGACAGCCGGCAGCAACAGTACCTGCAGCACACCAGCAGGAAGGAGATTCACTTTGGCAGCCCACAGCACCTGTGCTTTGCTGTCAGGCAGGAGCAGGTGATTCTTCAGAACTGCACGGAGGAAGGCCTGGCCATCCACCAGCAGCACTGGGACTTCCAGGAGAATGGGATGATTGTCCACATTCTTTCTGGGAAATGCATGGAAGCTGTGGTGCAAGAAAACAATAAAGATTTGTACCTGCGTCCGTGTGATGGAAAAGCCCGCCAGCAGTGGCGTTTTGACCAGATCA ATGCTGTGGATGAACGATGA.

[0216] Human GalNAc-T13 amino acid sequence:MLLRKRYRHRPCRLQFLLLLLMLGCVLMMVAMLHPPHHTLHQTVTAQASKHSPEARYRLDFGESQDWVLEAEDEGEEYSPLEGLPPFISLREDQLLVAVALPQARRNQSQGRRGGSYRLIKQPRRQDKEAPKRDWGADEDGEVSEEEELTPFSLDPRGLQEALSARIPLQRALPEVRHPLCLQQHPQDSLPTASVILCFHDEAWSTLLRTVHSILDTVPRAFLKEIILVDDLSQQGQLKSALSEYVARLEGVKLLRSNKRLGAIRARMLGATRATGDVLVFMDAHCECHPGWLEPLLSRIAGDRSRVVSPVIDVIDWKTFQYYPSKDLQRGVLDWKLDFHWEPLPEHVRKALQSPISPIRSPVVPGEVVAMDRIIYFQNTGAYDSLMSLRGGENLELSFKAWLCGGSVEILPCSRVGHIYQNQDSHSPLDQEATLRNRVRIAETWLGSFKETFYKHSPEAFSLSKAEKPDCMERLQLQRRLGCRTFHWFLANVYPELYPSEPRPSFSGKLHNTGLGLCADCQAEGDILGCPMVLAPCSDSRQQQYLQHTSRKEIHFGSPQHLCFAVRQEQVILQNCTEEGLAIHQQHWDFQENGMIVHILSGKCMEAVVQENNKDLYLRPCDGKARQQWRFDQINAVDER

[0217] The coding region for human polypeptide GalNAc-T14 has beensubmitted to GenBank/EBI Data Bank and assigned accession numberAJ505991.

[0218] Human GalNAc-T14 DNA sequence:ATGAGGAGATTTGTCTACTGCAAGGTGGTTCTAGCCACTTCGCTGATGTGGGTTCTTGTTGATGTCTTCTTACTGCTGTACTTCAGTGAATGTAACAAATGTGATGACAAGAAGGAGAGATCTCTGCTGCCTGCATTGAGGGCTGTTATTTCAAGAAACCAAGAAGGGCCAGGAGAAATGGGAAAAGCTGTGTTGATTCCTAAAGATGACCAGGAGAAAATGAAAGAGCTGTTTAAAATCAATCAGTTTAACCTTATGGCCAGTGATTTGATTGCCCTTAATAGAAGTCTGCCAGATGTAAGATTAGAAGGATGTAAGACAAAAGTCTACCCTGATGAACTTCCAAACACAAGTGTAGTCATTGTGTTTCATAATGAAGCTTGGAGCACTCTCCTTAGAACTGTTTACAGTGTGATAAATCGTTCCCCACACTATCTACTCTCAGAGGTCATCTTGGTAGATGATGCCAGTGAAAGAGATTTTCTCAAGTTGACATTAGAGAATTACGTGAAAAATTTAGAAGTGCCAGTAAAAATTATTAGGATGGAAGAACGCTCTGGGTTAATACGTGCCCGTCTTCGAGGAGCAGCTGCTTCAAAAGGGCAGGTCATAACTTTTCTTGATGCACACTGTGAATGCACGTTAGGATGGCTGGAGCCTTTGCTGGCAAGAATAAAGGAAGACAGGAAAACGGTTGTCTGCCCTATCATTGATGTGATTAGTGATGATACTTTTGAATATATGGCTGGGTCAGACATGACTTATGGGGGTTTTAACTGGAAACTGAATTTCCGCTGGTATCCTGTTCCCCAAAGAGAAATGGACAGGAGGAAAGGAGACAGAACATTACCTGTCAGGACCCCTACTATGGCTGGTGGCCTATTTTCTATTGACAGAAACTACTTTGAAGAGATAGGAACTTACGATGCAGGAATGGATATCTGGGGTGGAGAGAATCTTGAAATGTCTTTTAGGATTTGGCAATGTGGAGGCTCCTTGGAGATTGTTACTTGCTCCCATGTTGGTCATGTTTTTCGGAAGGCAACTCCATACACTTTTCCTGGTGGCACTGGTCATGTCATCAACAAGAACAACAGGAGACTGGCAGAAGTTTGGATGGATGAATTTAAAGATTTCTTCTACATCATATCCCCAGGTGTTGTCAAAGTGGATTATGGAGATGTGTCAGTCAGAAAAACACTAAGAGAAAATCTGAAGTGTAAGCCCTTTTCTTGGTACCTAGAAAACATCTATCCGGACTCCCAGATCCCAAGACGTTATTACTCACTTGGTGAGATAAGAAATGTTGAAACCAATCAGTGTTTAGACAACATGGGCCGCAAGGAAAATGAAAAAGTGGGTATATTCAACTGTCATGGTATGGGAGGAAATCAGGTATTTTCTTACACTGCTGACAAAGAAATCCGAACCGATGACTTGTGCTTGGATGTTTCTAGACTCAATGGACCTGTAATCATGTTAAAATGCCACCATATGAGAGGAAATCAGTTATGGGAATATGATGCTGAGAGACTCACGTTGCGACATGTTAACAGTAACCAATGTCTCGATGAACCTTCTGAAGAAGACAAAATGGTGCCTACAATGCAGGACTGTAGTGGAAGCAGATCCCAACAGTGGCTGCTAAGG AACATGACCTTGGGCACATGA

[0219] Human GalNAc-T14 amino acid sequence:MRRFVYCKVVLATSLMWVLVDVFLLLYFSECNKCDDKKERSLLPALRAVISRNQEGPGEMGKAVLIPKDDQEKMKELFKINQFNLMASDLIALNRSLPDVRLEGCKTKVYPDELPNTSVVIVFHNEAWSTLLRTVYSVINRSPHYLLSEVILVDDASERDFLKLTLENYVKNLEVPVKIIRMEERSGLIRARLRGAAASKGQVITFLDAHCECTLGWLEPLLARIKEDRKTVVCPIIDVISDDTFEYMAGSDMTYGGFNWKLNFRWYPVPQREMDRRKGDRTLPVRTPTMAGGLFSIDRNYFEEIGTYDAGMDIWGGENLEMSFRIWQCGGSLEIVTCSHVGHVFRKATPYTFPGGTGHVINKNNRRLAEVWMDEFKDFFYIISPGVVKVDYGDVSVRKTLRENLKCKPFSWYLENIYPDSQIPRRYYSLGEIRNVETNQCLDNMGRKENEKVGIFNCHGMGGNQVFSYTADKEIRTDDLCLDVSRLNGPVIMLKCHHMRGNQLWEYDAERLTLRHVNSNQCLDEPSEEDKMVPTMQDCSGSRSQQWLLR NMTLGT

[0220] The coding region for human polypeptide GalNAc-T15 has beensubmitted to GenBank/EBI Data Bank and assigned accession number Y09324.

[0221] Human GalNAc-T15 DNA sequence:ATGCGGCGCCTGACTCGTCGGCTGGTTCTGCCAGTCTTCGGGGTGCTCTGGATCACGGTGCTGCTGTTCTTCTGGGTAACCAAGAGGAAGTTGGAGGTGCCGACGGGACCTGAAGTGCAGACCCCTAAGCCTTCGGACGCTGACTGGGACGACCTGTGGGACCAGTTTGATGAGCGGCGGTATCTGAATGCCAAAAAGTGGCGCGTTGGTGACGACCCCTATAAGCTGTATGCTTTCAACCAGCGGGAGAGTGAGCGGATCTCCAGCAATCGGGCCATCCCGGACACTCGCCATCTGAGATGCACACTGCTGGTGTATTGCACGGACCTTCCACCCACTAGCATCATCATCACCTTCCACAACGAAGCCCGCTCCACGCTGCTCAGGACCATCCGCAGTGTATTAAACCGCACCCCTACGCATCTGATCCGGGAAATCATATTAGTGGATGACTTCAGCAATGACCCTGATGACTGTAAACAGCTCATCAAATTGCCCAAGGTGAAATGCTTGCGCAATAATGAACGGCAAGGTCTGGTCCGGTCCCGGATTCGGGGCGCTGACATCGCCCAGGGCACCACTCTGACTTTCCTCGACAGCCACTGTGAGGTGAACAGGGACTGGCTCCAGCCTCTGTTGCACAGGGTCAAAGAAGACTACACGCGGGTGGTGTGCCCTGTGATCGATATCATTAACCTGGACACCTTCACCTACATCGAGTCTGCCTCGGAGCTCAGAGGGGGGTTTGACTGGAGCCTCCACTTCCAGTGGGAGCAGCTCTCCCCAGAGCAGAAGCTCGGCGCCTGGACCCCACGGAAGCCCATCAGGACTCCTATCATAGCTGGAGGGCTCTTCGTGATCGACAAAGCTTGGTTTGATTACCTGGGGAAATATGATATGGACATGGACATCTGGGGTGGGGAGAACTTTGAAATCTCCTTCCGAGTGTGGATGTGCGGGGGCAGCCTAGAGATCGTCCCCTGCAGCCGAGTGGGGCACGTCTTCCGGAAGAAGCACCCCTACGTTTTCCCTGATGGAAATGCCAACACGTATATAAAGAACACCAAGCGGACAGCTGAAGTGTGGATGGATGAATACAAGCAATACTATTACGCTGCCCGGCCATTCGCCCTGGAGAGGCCCTTCGGGAATGTTGAGAGCAGATTGGACCTGAGGAAGAATCTGCGCTGCCAGAGCTTCAAGTGGTACCTGGAGAATATCTACCCTGAACTCAGCATCCCCAAGGAGTCCTCCATCCAGAAGGGCAATATCCGACAGAGACAGAAGTGCCTGGAATCTCAAAGGCAGAACAACCAAGAAACCCCAAACCTAAAGTTGAGCCCCTGTGCCAAGGTCAAAGGCGAAGATGCAAAGTCCCAGGTATGGGCCTTCACATACACCCAGCAGATCCTCCAGGAGGAGCTGTGCCTGTCAGTCATCACCTTGTTCCCTGGCGCCCCAGTGGTTCTTGTCCTTTGCAAGAATGGAGATGACCGACAGCAATGGACCAAAACTGGTTCCCACATCGAGCACATAGCATCCCACCTCTGCCTCGATACAGATATGTTCGGTGATGGCACCGAGAACGGCAAGGAAATCGTCGTCAACCCATGTGAGTCCTCACTCATGAGCCAGCACTGGGACATGGTGAGCTCTTGAGGACCCCTGCCAGAAGCAGCAAGGGCCATGGGGTGGTGCTTCCCTGGACCAGAACAGACTGGAAACTGGGCAGCAAGCAGCCTGCAACCACCTCAGACATCCTGGACTGGGAGGTGGAGGCAGAGCCCCCCAGGACAGGAGCAACTGTCTCAGGGAGGACAGAGGAAAACATCACAAGCCAATGGGGCTCAAAGACAAATCCCACATGTTCTCAAGGCCGTTAAGTTCCAGTCCTGGCCAG TCATTCCCTGA

[0222] Human GalNAc-T15 amino acid sequence:MRRLTRRLVLPVFGVLWITVLLFFWVTKRKLEVPTGPEVQTPKPSDADWDDLWDQFDERRYLNAKKWRVGDDPYKLYAFNQRESERISSNRAIPDTRHLRCTLLVYCTDLPPTSIIITFHNEARSTLLRTIRSVLNRTPTHLIREIILVDDFSNDPDDCKQLIKLPKVKCLRNNERQGLVRSRIRGADIAQGTTLTFLDSHCEVNRDWLQPLLHRVKEDYTRVVCPVIDIINLDTFTYIESASELRGGFDWSLHFQWEQLSPEQKLGAWTPRKPIRTPIIAGGLFVIDKAWFDYLGKYDMDMDIWGGENFEISFRVWMCGGSLEIVPCSRVGHVFRKKHPYVFPDGNANTYIKNTKRTAEVWMDEYKQYYYAARPFALERPFGNVESRLDLRKNLRCQSFKWYLENIYPELSIPKESSIQKGNIRQRQKCLESQRQNNQETPNLKLSPCAKVKGEDAKSQVWAFTYTQQILQEELCLSVITLFPGAPVVLVLCKNGDDRQQWTKTGSHLEHIASHLCLDTDMFGDGTENGKEIVVNPCESSLMSQHWDMV SS

[0223] The coding region for human polypeptide GalNAc-T16 has beensubmitted to GenBank/EBI Data Bank and assigned accession numberAJ505951.

[0224] Human GalNAc-T16 DNA sequence:ATGAGGAAGATCCGCGCCAATGCCATCGCCATCCTGACCGTAGCCTGGATCCTGGGCACTTTCTACTACTTATGGCAGGACAACCGAGCCCACGCAGCATCCTCCGGCGGCCGGGGCGCGCAGAGGGCAGGCAGGAGGTCGGAGCAGCTCCGCGAGGACCGCACCATCCCGCTCATTGTGACAGGAACTCCCTCGAAAGGCTTTGATGAGAAGGCCTACCTGTCGGCCAAGCAGCTGAAGGCTGGAGAGGACCCCTACAGACAGCACGCCTTCAACCAGCTGGAGAGTGACAAGCTGAGCCCAGACCGGCCCATCCGGGACACCCGCCATTACAGCTGCCCATCTGTGTCCTACTCCTCGGACCTGCCAGCCACCAGCGTCATCATCACCTTCCACAATGAGGCCCGTTCCACCCTGCTGCGCACAGTGAAGAGTGTCCTGAACCGAACTCCTGCCAACTTGATCCAGGAGATCATTTTAGTGGATGACTTCAGCTCAGATCCGGAAGACTGTCTACTCCTGACCAGGATCCCCAAGGTCAAGTGCCTGCGCAATGATCGGCGGGAAGGGCTGATCCGGTCCCGAGTGCGTGGGGCGGACGTGGCTGCAGCTACCGTTCTCACCTTTCTGGATAGCCACTGCGAAGTGAACACCGAGTGGCTGCCGCCCATGCTGCAGCGGGTGAAGGAGGACCACACCCGCGTGGTGAGTCCCATCATTGATGTCATCAGTCTGGATAATTTTGCCTACCTTGCAGCATCTGCTGACCTTCGTGGAGGGTTCGACTGGAGCCTGCATTTCAAGTGGGAGCAGATCCCTCTTGAGCAGAAGATGACCCGGACAGACCCCACCAGGCCCATAAGGACGCCTGTCATAGCTGGAGGAATCTTCGTGATCGACAAGTCCTGGTTTAACCACTTGGGAAAGTATGATGCCCAGATGGACATCTGGGGGGGAGAGAATTTTGAGCTCTCCTTCAGGGTGTGGATGTGTGGTGGCAGTCTGGAGATCGTCCCCTGCAGCCGGGTGGGCCATGTCTTCAGGAAACGGCACCCCTACAACTTCCCTGAGGGTAATGCCCTCACCTACATCAGGAATACTAAGCGCACTGCAGAAGTGTGGATGGATGAATACAAGCAATACTACTATGAGGCCCGGCCCTCGGCCATCGGGAAGGCCTTCGGCAGTGTGGCTACGCGGATAGAGCAGAGGAAGAAGATGAACTGCAAGTCCTTCCGCTGGTACCTGGAGAACGTCTACCCAGAGCTCACGGTCCCCGTGAAGGAAGCACTCCCCGGCATCATTAAGCAGGGGGTGAACTGCTTAGAATCTCAGGGCCAGAACACAGCTGGTGACTTCCTGCTTGGAATGGGGATCTGCAGAGGGTCTGCCAAGAACCCGCAGCCCGCCCAGGCATGGCTGTTCAGTGACCACCTCATCCAGCAGCAGGGGAAGTGCCTGGCTGCCACCTCCACCTTAATGTCCTCCCCTGGATCCCCAGTCATACTGCAGATGTGCAACCCTAGAGAAGGCAAGCAGAAATGGAGGAGAAAAGGATCTTTCATCCAGCATTCAGTCAGTGGCCTCTGCCTGGAGACAAAGCCTGCCCAGCTGGTGACCAGCAAGTGTCAGGCTGACGCCCAGGCCCAGCAGTGGCAGCTGTTGCCACACACATGA

[0225] Human GalNAc-T16 amino acid sequence:MRKIRANAIAILTVAWILGTFYYLWQDNRAHAASSGGRGAQRAGRRSEQLREDRTIPLIVTGTPSKGFDEKAYLSAKQLKAGEDPYRQHAFNQLESDKLSPDRPIRDTRHYSCPSVSYSSDLPATSVIITFHNEARSTLLRTVKSVLNRTPANLIQEIILVDDFSSDPEDCLLLTRIPKVKCLRNDRREGLIRSRVRGADVAAATVLTFLDSHCEVNTEWLPPMLQRVKEDIITRVVSPIIDVISLDNFAYLAASADLRGGFDWSLHFKWEQIPLEQKMTRTDPTRPIRTPVIAGGIFVIDKSWFNIILGKYDAQMDIWGGENFELSFRVWMCGGSLEIVPCSRVGHVFRKRHPYNFPEGNALTYIRNTKRTAEVWMDEYKQYYYEARPSAIGKAFGSVATRIEQRKKMNCKSFRWYLENVYPELTVPVKEALPGIIKQGVNCLESQGQNTAGDFLLGMGICRGSAKNPQPAQAWLFSDHLIQQQGKCLAATSTLMSSPGSPVILQMCNPREGKQKWRRKGSFIQHSVSGLCLETKPAQLVTSKCQADAQ AQQWQLLPHT

[0226] Additional homologous polypeptide GalNAc-transferase genes havebeen identified and cloning and expression are in progress, and itfollows from the descriptions that similar methods as outlined abovewill yield soluble secreted proteins for study. Expression constructsmay have immunoaffinity tags or purification tags at the N-terminaland/or C-terminal region. These may include myc, FLAG, HIS, GST, andother (Stratagene, Qiagen, Amersham Biosciences).

[0227] Soluble secreted expression constructs of GalNAc-transferaselectin domains were prepared from the GalNAc-transferase expressionconstructs described above by PCR using primer pairs as listed Table Vbelow. GalNAc-Ti lectin domain: T1LECFOR:5′-CAAGGAAGCTTATGGGGATATATCGTCAAGAG-3′ T1LECREV:5′-GCAAGCTCGAGGCGGCCGCTCAGAATATTTCTGGAAGGGTGAC-3′ GalNAc-T2 lectindomain: T2LECFOR: 5′-CAAGGAACCTTCTTATGCAAATATTCAGAGCAGATTG-3′ T2LECREV:5′-GCAAGCTCGAGGCGGCCGCCTACTGCTGCAGGTTGAGC-3′ GalNAc-T3 lectin domain:T3LECFOR: 5′-CAAGGAAGCTTCATTTGGTGATCTTTCAAAAAGATTT-3′ T3LECREV:5′-GCAACCTCGACGCGGCCGCAGGAACACTTAATCATTTTGG-3′ GalNAc-T4 lectin domain:T4LECFOR: 5′-ACAAAAGAAGCTTATGGTGATATTTCTC-3′ EBHC307:5′-AGCGGATCCGACGAAGTGCTGTTGTGCT-3′ GalNAc-T5 lectin domain: TSLECFOR:5′-CAAGGAAGCTTTAGATGTTGGCAACCTCACCCAGC-3′ T5LECREV:5′-GCAAGCTCGAGGCGGCCGCAAGCATCAGTTACACTTCAGGCTTC-3′ GalNAc-T6 lectindomain: TELECFOR: 5′-CAAGGAAGCTTCCTTCGGTGACATTTCGCAACG-3′ TGLECREV:5′-GCAAGCTCGAGGCGGCCGCTGGGTCCTAGACAAAGAGCC-3′ GalNAc-T7 lectin domain:T7LECFOR: 5′-AGAAAAGAAGCTTATGGGGATATATCGGAGCTG-3′ T7LECREV:5′-GCAAGCTCGAGGCGGCCGCTCTCTAAACACTATGGATGTTATTC-3′ GalNAc-T8 lectindomain: T8LECFOR: 5′-CAAGGAAGCTTTTGGAGACGTTTCTTCCAGAATG-3′ T8LECREV:5′-GCAAGCTCGAGGCGGCCGCTCACTGCCTGTTGCTCTGACCCC-3′ GalNAc-T9 lectm domain:T9LECFOR: 5′-CAAGGAAGCTTTCCGGGACGTGTCTCAGAGACTG-3′ T9LECREV:5′-GCAAGCTCGAGGCGGCCGCTCAGTGCCGTGCGTGTTTGATTCC-3′ GalNAc-T10 lectindomain: T10LECFOR: 5′-CAAGGAAGCTTCCGCTGGGGATGTCGCAGTCCAG-3′ T10LECREV:5′-GCAAGCTCGAGGCGCCCCCTCAGTTCCTATTGAATTTTTCC-3′ GalNAc-T11 lectindomain: T11LECFOR: 5′-CAACCAAGCTTGCAATATCAGTGAGCGTGTGG-3′ T11LECREV:5′-GCAAGCTCGAGGCGGCCGCCCACCTTAACCTTCCAAATGC-3′ GalNAc-T12 lectin domain:T12LECFOR: 5′-CAAGGAAGCTTGGGATGTGACAGAGAGGAAG-3′ T12LECREV:5′-GCAAGCTCGAGGCGGCCGCTCATAACATCCCCTCTTTGAAGAACC-3′ GalNAc-T13 lectindomain: T13LECFOR: 5′-CAAGGAAGCTTCTGAGAAGCCAGACTGCATGG-3′ T13LECREV:5′-GCAAGCTCCAGGCCCCCGCTCATCGTTCATCCACACCATTC-3′ Ga1NAc-T14 lectindomain: T14LECFOR: 5′-CAAGGAAGCTTATGCAGATGTGTCAGTCAGAAAAAC-3′ T14LECREV:5′-GCAAGCTCGAGGCGGCCGCTCATGTGCCCAAGGTCATGTTCC-3′ GaLNAc-T15 lectindomain: T15LECFOR: 5′-CAACGAACCTTTCCGCAATGTTGAGAGCAGATTG-3′ T15LECREV:5′-GCAAGCTCCAGCCGGCCGCTCAAGAACTCACCATCTCCCAGTG-3′ GalNAc-T16 lectmdomain: T16LECFOR: 5′-CAAGGAAGCTTGCAGTGTGGCTACGCGGATAGAGCAGAGG-3′T16LECREV: 5′-GCAAGCTCGAGGCCGCCGCTCATGTGTGTGGCAACAGCTCCC-3′

[0228] PCR amplifications were performed with 10 ng GalNAc-transferaseplasmid DNA as template and High Fidelity PCR kit (Roche) withconditions recommended by the manufacturer. Amplified products weredigested with HindIII and XhoI and inserted into the HindIII/XhoI siteof pBKS-HistagI. All constructs were fully sequenced. Tagged lectindomain constructs were excised with NotI and sub-cloned into the NotIsite of pAcGP67-A Baculo expression vector.

[0229] The exact borders of the lectin domains and the catalytic unitshave not been defined, but multiple sequence alignment analysis (FIG. 4)was used to predict the most likely borders and these were used fordesign of PCR primers as listed in Table V. DNA and amino acid sequencesof preferred constructs of GalNAc-transferase lectin domains and theirconstruct design include the following (Table VI):

[0230] Table VI: DNA and amino acid sequences of GalNAc-transferaselectin domains.

[0231] GalNAc-T1 Lectin Domain:

[0232] The lectin domain polypeptide sequence comprises amino acidresidues 393-559 of GALANT1 (GALNT1 nucleotide sequence accession numberis AJ505952)

[0233] T1 LECTIN DNA Sequence:AAAGAAGCTTATGGAGATATATCGTCAAGAGTTGGTCTAAGACACAAACTACAATGCAAACCTTTTTCCTGGTACCTAGAGAATATATATCCTGATTCTCAAATTCCACGTCACTATTTCTCATTGGGAGAGATACGAAATGTGGAAACGAATCAGTGTCTAGATAACATGGCTAGAAAAGAGAATGAAAAAGTTGGAATTTTTAATTGCCATGGTATGGGGGGTAATCAGGTTTTCTCTTATACTGCCAACAAAGAAATTAGAACAGATGACCTTTGCTTGGATGTTTCCAAACTTAATGGCCCAGTTACAATGCTCAAATGCCACCACCTAAAAGGCAACCAACTCTGGGAGTATGACCCAGTGAAATTAACCCTGCAGCATGTGAACAGTAATCAGTGCCTGGATAAAGCCACAGAAGAGGATAGCCAGGTGCCCAGCATTAGAGACTGCAATGGAAGTCGGTCCCAGCAGTGGCTTCTTCGAAACGTCACCCTTCC AGAAATATTC TGA- stop

[0234] T1 LECTIN Amino Acid Sequence:YGDISSRVGLRTIKLQCKPFSWYLENIYPDSQIPRHYFSLGEIRNVETNQCLDNMARKENEKVGIFNCHGMGGNQVFSYTANKEIRTDDLCLDVSKLNGPVTMLKCHHLKGNQLWEYDPVKLTLQHVNSNQCLDKATEEDSQVPSIRDCN GSRSQQWLLRNVTLPEIF*

[0235] GalNAc-T2 Lectin Domain:

[0236] The lectin domain polypeptide sequence comprises amino acidresidues 408-571 of GALNT2 (GALNT2 nucleotide sequence accession numberis X85019).

[0237] T2 LECTIN DNA sequence:TATCCAGAGTTAAGGGTTCCAGACCATCAGGATATAGCTTTTGGGGCCTTGCAGCAGGGAACTAACTGCCTCGACACTTTGGGACACTTTGCTGATGGTGTGGTTGGAGTTTATGAATGTCACAATGCTGGGGGAAACCAGGAATGGGCCTTGACGAAGGAGAAGTCGGTGAAGCACATGGATTTGTGCCTTACTGTGGTGGACCGGGCACCGGGCTCTCTTATAAAGCTGCAGGGCTGCCGAGAAAATGACAGCAGACAGAAATGGGAACAGATCGAGGGCAACTCCAAGCTGAGGCACGTGGGCAGGAACCTGTGCCTGGACAGTCGCACGGCCAAGAGCGGGGGCCTAAGCGTGGAGGTGTGTGGCCCGGCCCTTTCGCAGCAGTGGAAGTTCACGC TCAACCTGCAGCAGTAG -Stop

[0238] T2 LECTIN Amino Acid Sequence:YPELRVPDHQDIAFGALQQGTNCLDTLGHFADGVVGVYECHNAGGNQEWALTKEKSVKHMDLCLTVVDRAPGSLIKLQGCRENDSRQKWEQIEGNSKLRHVGSNLCLDSRTAKSGGLSVEVCGPALSQQWKFTLNLQQ*

[0239] GalNAc-T3 Lectin Domain:

[0240] The lectin domain polypeptide sequence comprises amino acidresidues 467-633 of GALNT3 (GALNT3 nucleotide sequence accession numberis AJ505954).

[0241] T3 LECTIN DNA sequence:TCATTTGGTGATCTTTCAAAAAGATTTGAAATAAAACACCGTCTTCGGTGTAAAAATTTTACATGGTATCTGAACAACATTTATCCAGAGGTGTATGTGCCAGACCTTAATCCTGTTATATCTGGATACATTAAAAGCGTTGGTCAGCCTCTATGTCTGGATGTTGGAGAAAACAATCAAGGAGGCAAACCATTAATTATGTATACATGTCATGGACTTGGGGGAAACCAGTACTTTGAATACTCTGCTCAACATGAAATTCGGCACAACATCCAGAAGGAATTATGTCTTCATGCTGCTCAAGGTCTCGTTCAGCTGAAGGCATGTACCTACAAAGGTCACAAGACAGTTGTCACTGGAGAGCAGATATGGGAGATCCAGAAGGATCAACTTCTATACAATCCATTCTTAAAAATGTGCCTTTCAGCAAATGGAGAGCATCCAAGTTTAGTGTCATGCAACCCATCAGATCCACTCCAAAAATGGATACTTAGCCAAAA TGATTAA-stop

[0242] T3 LECTIN Amino Acid Sequence:FGDLSKRFEIKHRLRCKNFTWYLNNIYPEVYVPDLNPVISGYIKSVGQPLCLDVGENNQGGKPLIMYTCHGLGGNQYFEYSAQHEIRHNIQKELCLHAAQGLVQLKACTYKGHKTVVTGEQIWEIQKDQLLYNPFLKMCLSANGEIIPSL VSCNPSDPLQKWILSQND*

[0243] GalNAc-T4 Lectin Domain:

[0244] The lectin domain polypeptide sequence comprises amino acidresidues 405-578 of GALNT4 (GALNT4 nucleotide sequence accession numberis Y08564).

[0245] T4 LECTIN DNA sequence:GAGGATAGACCAGGCTGGCATGGGGCTATTCGCAGTAGAGGGATCTCGTCTGAATGTTTAGATTATAATTCTCCTGACAACAACCCCACAGGTGCTAACCTTTCACTGTTTGGATGCCATGGTCAAGGAGGCAATCAATTCTTTGAATATACTTCAAACAAAGAAATAAGGTTTAATTCTGTGACAGAGTTATGTGCAGAGGTACCTGAGCAAAAAAATTATGTGGGAATGCAAAATTGTCCCAAAGATGGGTTCCCTGTACCAGCAAACATTATTTGGCATTTTAAAGAAGATGGAACTATTTTTCACCCACACTCAGGACTGTGTCTTAGTGCTTATCGGACACCGGAGGGCCGACCTGATGTACAAATGAGAACTTGTGATGCTCTAGATAAAAATC AAATTTGGAGTTTTGAGAAATAG-stop

[0246] T4 LECTIN Amino Acid SequenceAYGDISERKLLRERLRCKSFDWYLKNVFPNLHVPEDRPGWHGAIRSRGISSECLDYNSPDNNPTGANLSLFGCHGQGGNQFFEYTSNKEIRFNSVTELCAEVPEQKNYVGMQNCPKDGFPVPANIIWHFKEDGTIFHPHSGLCLSAYRTPEGRPDVQMRTCDALDKNQIWSFEK*

[0247] GalNAc-T5 Lectin Domain:

[0248] The lectin domain polypeptide sequence comprises amino acidresidues 486-653 of GALTN5 (GALTN5 nucleotide sequence accession numberis AJ505956).

[0249] T5 LECTIN DNA SequenceTTAGATGTTGGCAACCTCACCCAGCAAAGGGAGCTGCGAAAGAAACTGAAGTGCAAAAGTTTCAAATGGTACTTGGAGAATGTCTTTCCTGACTTAAGGGCTCCCATTGTGAGAGCTAGTGGTGTGCTTATTAATGTGGCTTTGGGTAAATGCATTTCCATTGAAAACACTACAGTCATTCTGGAAGACTGCGATGGGAGCAAAGAGCTTCAACAATTTAATTACACCTGGTTAAGACTTATTAAATGTGGAGAATGGTGTATAGCCCCCATCCCTGATAAAGGAGCCGTAAGGCTGCACCCTTGTGATAACAGAAACAAAGGGCTAAAATGGCTGCATAAATCAACATCAGTCTTTCATCCAGAACTGGTGAATCACATTGTTTTTGAAAACAATCAGCAATTATTATGCTTGGAAGGAAATTTTTCTCAAAAGATCCTGAAAGTAGCTGCCTGTGACCCAGTGAAGCCATATCAAAAGTGGAAATTTGAAAAATATTA TGAAGCC TGA-stop

[0250] T5 LECTIN Amino Acid SequenceDVGNLTQQRELRKKLKCKSFKWYLENVFPDLRAPIVRASGVLINVALGKCISIENTTVILEDCDGSKELQQFNYTWLRLIKCGEWCIAPIPDKGAVRLHPCDNRNKGLKWLHKSTSVFHPELVNHIVFENNQQLLCLEGNFSQKILKVAA CDPVKPYQKWKFEKYYEA*

[0251] GalNAc-T6 Lectin Domain:

[0252] The lectin polypeptide sequence comprises amino acid residues458-622 of GALNT6 (GALNT6 nucleotide sequence accession number isAJ133523).

[0253] T6 LECTIN DNA Sequence:TCCTTCGGTGACATTTCGGAACGACTGCAGCTGAGGGAACAACTGCACTGTCACAACTTTTCCTGGTACCTGCACAATGTCTACCCAGAGATGTTTGTTCCTGACCTGACGCCCACCTTCTATGGTGCCATCAAGAACCTCGGCACCAACCAATGCCTGGATGTGGGTGAGAACAACCGCGGGGGGAAGCCCCTCATCATGTACTCCTGCCACGGCCTTGGCGGCAACCAGTACTTTGAGTACACAACTCAGAGGGACCTTCGCCACAACATCGCAAAGCAGCTGTGTCTACATGTCAGCAAGGGTGCTCTGGGCCTTGGGAGCTGTCACTTCACTGGCAAGAATAGCCAGGTCCCCAAGGACGAGGAATGGGAATTGGCCCAGGATCAGCTCATCAGGAACTCAGGATCTGGTACCTGCCTGACATCCCAGGACAAAAAGCCAGCCATGGCCCCCTGCAATCCCAGTGACCCCCATCAGTTGTGGCTCTTTGTC TAG- stop

[0254] T6 LECTIN Amino Acid Sequence:SFGDISERLQLREQLHCHNFSWYLHNVYPEMFVPDLTPTEYGAIKNLGTNQCLDVGENNRGGKPLIMYSCHGLGGNQYFEYTTQRDLRHNIAKQLCLHVSKGALGLGSCHFTGKNSQVPKDEEWELAQDQLIRNSGSGTCLTSQDKKPAM APCNPSDPHQLWLFV*

[0255] GalNAc-T7 Lectin Domain:

[0256] The lectin domain polypeptide sequence comprises amino acidresidues 492-657 of GALNT7 (GALNT7 nucleotide sequence accession numberis AJ505958).

[0257] T7 LECTIN DNA Sequence:TATGGGGATATATCGGAGCTGAAAAAATTTCGAGAAGATCACAACTGCCAAAGTTTTAAGTGGTTCATGGAAGAAATAGCTTATGATATCACCTCACACTACCCTTTGCCACCCAAAAATGTTGACTGGGGAGAAATCAGAGGCTTCGAAACTGCTTACTGCATTGATAGCATGGGAAAAACAAATGGAGGCTTTGTTGAACTAGGACCCTGCCACAGGATGGGAGGGAATCAGCTTTTCAGAATCAATGAAGCAAATCAACTCATGCAGTATGACCAGTGTTTGACAAAGGGAGCTGATGGATCAAAAGTTATGATTACACACTGTAATCTAAATGAATTTAAGGAATGGCAGTACTTCAAGAACCTGCACAGATTTACTCATATTCCTTCAGGAAAGTGTTTAGATCGCTCAGAGGTCCTGCATCAAGTATTCATCTCCAATTGTGACTCCAGTAAAACGACTCAAAAATGGGAAATGAATAACATCCATAGTGTT TAG-stop

[0258] T7 LECTIN Amino Acid Sequence:YGDISELKKFREDHNCQSFKWFMEEIAYDITSHYPLPPKNVDWGEIRGFETAYCIDSMGKTNGGFVELGPCHRMGGNQLFRINEANQLMQYDQCLTKGADGSKVMITHCNLNEFKEWQYFKNLHRFTHIPSGKCLDRSEVLHQVFISNCD SSKTTQKWEMNNIHSV*

[0259] GalNAc-T8 Lectin Domain:

[0260] The lectin domain polypeptide sequence comprises amino acidresidues 459-637 of GALNT8 (GALNT8 nucleotide sequence accession numberis AJ505959).

[0261] T8 LECTIN DNA Sequence:GACGTTTCTTCCAGAATGGCACTCCGGGAAAAACTGAAATGTAAAACTTTTGACTGGTACCTGAAAAATGTTTATCCACTCTTGAAGCCACTCCACACCATCGTGGGCTATGGAAGAATGAAAAACCTATTGGATGAAAATGTCTGCTTGGATCAGGGACCCGTTCCAGGCAACACCCCCATCATGTATTACTGCCATGAATTCAGCTCACAGAATGTCTACTATCACCTAACTGGGGAGCTCTATGTGGGACAACTGATTGCAGAGGCCAGTGCTAGTGATCGCTGCCTGACAGACCCTGGCAAGGCGGAGAAGCCCACCTTAGAACCATGCTCCAAGGCAGCTAAGAATAGACTGCATATATATTGGGATTTTAAACCGGGAGGAGCTGTCATAAACAGAGATACCAAGCGGTGTCTGGAGATGAAGAAGGATCTTTTGGGTAGCCACGTGCTTGTGCTCCAGACCTGTAGCACGCAAGTGTGGGAAATCCAGCACACTGTCAGAGACTGGGGTCAGACCAACAGCCAGTGA

[0262] T8 LECTIN Amino Acid Sequence:FGDVSSRMALREKLKCKTFDWYLKNVYPLLKPLHTIVGYGRMKNLLDENVCLDQGPVPGNTPIMYYCHEFSSQNVYYHLTGELYVGQLIAEASASDRCLTDPGKAEKPTLEPCSKAAKNRLHIYWDFKPGGAVINRDTKRCLEMKKDLLGSHVLVLQTCSTQVWEIQHTVRDWGQTNSQ

[0263] GalNAc-T9 Lectin Domain:

[0264] The lectin domain polypeptide sequence comprises amino acidresidues 427-603 of GALNT9 (GALNT9 nucleotide sequence accession numberis AJ505960).

[0265] T9 LECTIN DNA Sequence:TTCGGGGACGTGTCTGAGAGGCTGGCCCTGCGTCAGAGGCTGAAGTGTCGCAGCTTCAAGTGGTACCTGGAGAACGTGTACCCGGAGATGAGGGTCTACAACAACACCCTCACGTACGGAGAGGTGAGAAACAGCAAAGCCAGTGCCTACTGTCTGGACCAGGGAGCGGAGGACGGCGACCGGGCGATCCTCTACCCCTGCCACGGGATGTCCTCCCAGCTGGTGCGGTACAGCGCTGACGGCCTGCTGCAGCTGGGGCCTCTGGGCTCCACAGCCTTCTTGCCTGACTCCAAGTGTCTGGTGGATGACGGCACGGGCCGCATGCCCACCCTGAAGAGGTGTGAGGATGTGGCGCGGCCAACACAGCGGCTGTGGGACTTCACCCAGAGTGGCCCCATTGTGAGCCGGGCCACGGGCCGCTGCCTGGAGGTGGAGATGTCCAAAGATGCCAACTTTGGGCTCCGGCTGGTGGTACAGAGGTGCTCGGGGCAGAAGTGGATGATCAGAAACTGGATCAAACACGCACGGCAC TGA-stop

[0266] T9 LECTIN Amino Acid Sequence:FGDVSERLALRQRLKCRSFKWYLENVYPEMRVYNNTLTYGEVRNSKASAYCLDQGAEDGDRAILYPCHGMSSQLVRYSADGLLQLGPLGSTAFLPDSKCLVDDGTGRMPTLKRCEDVARPTQRLWDFTQSGPIVSRATGRCLEVEMSKDANFGLRLVVQRCSGQKWMIRNWIKHARH*

[0267] GalNAc-T10 Lectin Domain:

[0268] The lectin domain polypeptide sequence comprises amino acidresidues 417-603 of GALNT10 (GALNT10 nucleotide sequence accessionnumber is AJ505950).

[0269] T1O LECTIN DNA Sequence:GCTGGGGATGTCGCAGTCCAGAAAAAGCTCCGCAGCTCCCTTAACTGCAAGAGTTTCAAGTGGTTTATGACGAAGATAGCCTGGGACCTGCCCAAATTCTACCCACCCGTGGAGCCCCCGGCTGCAGCTTGGGGGGAGATCCGAAATGTGGGCACAGGGCTGTGTGCAGACACAAAGCACGGGGCCTTGGGCTCCCCACTAAGGCTAGAGGGCTGCGTCCGAGGCCGTGGGGAGGCTGCCTGGAACAACATGCAGGTATTCACCTTCACCTGGAGAGAGGACATCCGGCCTGGAGACCCCCAGCACACCAAGAAGTTCTGCTTTGATGCCATTTCCCACACCAGCCCTGTCACGCTGTACGACTGCCACAGCATGAAGGGCAACCAGCTGTGGAAATACCGCAAAGACAAGACCCTGTACCACCCTGTCAGTGGCAGCTGCATGGACTGCAGTGAAAGTGACCATAGGATCTTCATGAACACCTGCAACCCATCCTCTCTCACCCAGCAGTGGCTGTTTGAACACACCAACTCAACAGTCTTGGAAAAAT TCAATAGGAACTGA

[0270] T10 LECTIN Amino Acid Sequence:AGDVAVQKKLRSSLNCKSFKWFMTKJAWDLPKFYPPVEPPAAAWGEIRNVGTGLCADTKHGALGSPLRLEGCVRGRGEAAWNNMQVFTFTWREDIRPGDPQHTKKFCFDAISHTSPVTLYDCITSMKGNQLWKYRKDKTLYHPVSGSCMDCSESDHRIFMNTCNPSSLTQQWLFEHTNSTVLEKFNRN*

[0271] GalNAc-T11 lectin domain:

[0272] The lectin domain polypeptide sequence comprises amino acidresidues 492-608 of GALNT11 (GALNT11 nucleotide sequence accessionnumber is Y12434).

[0273] T11 LECTIN DNA sequence:TGCAATATCAGTGAGCGTGTGGAACTGAGAAAGAAGTTGGGCTGTAAATCATTTAAATGGTATTTGGATAATGTATACCCAGAGATGCAGATATCTGGGTCCCACGCCAAACCCCAACAACCCATTTTTGTCAATAGAGGGCCAAAACGACCCAAAGTCCTTCAACGTGGAAGGCTCTATCACCTCCAGACCAACAAATGCCTGGTGGCCCAGGGCCGCCCAAGTCAGAAGGGAGGTCTCGTGGTGCTTAAGGCCTGTGACTACAGTGACCCAAATCAGATCTGGATCTATAATGAAGAGCATGAATTGGTTTTAAATAGTCTCCTTTGTCTAGATATGTCAGAGACTCGCTCATCAGACCCGCCACGGCTCATGAAATGCCACGGGTCAGGAGGATCCCAGCAGTGGACCTTTGGGAAAAACAATCGGCTATACCAGGTGTCGGTTGGACAGTGCCTGAGAGCAGTGGATCCCCTGGGTCAGAAGGGCTCTGTCGCCATGGCGATCTGCGATGGCTCCTCTTCACAGCAGTGGCATTTGGAAGGTTAA

[0274] T11 LECTIN Amino Acid Sequence:NISERVELRKKLGCKSFKWYLDNVYPEMQISGSHAKPQQPIFVNRGPKRPKVLQRGRLYHLQTNKCLVAQGRPSQKGGLVVLKACDYSDPNQIWIYNEEHELVLNSLLCLDMSETRSSDPPRLMKCHGSGGSQQWTFGKNNRLYQVSVGQCLRAVDPLGQKGSVAMAICDGSSSQQWHLEG*

[0275] GalNAc-T12 Lectin Domain:

[0276] The lectin domain polypeptide sequence comprises amino acidresidues 428-581 of GALN T12 (GALNT12 nucleotide sequence accessionnumber is AJ505963). T12 LECTIN DNA sequence:TGGGATGTGACAGAGAGGAAGCAGCTCCGGGACAAGCTCCAGTGTAAAGACTTCAAGTGGTTCTTGGAGACTGTGTATCCAGAACTGCATGTGCCTGAGGACAGGCCTGGCTTCTTCGGGATGCTCCAGAACAAAGGACTAACAGACTACTGCTTTGACTATAACCCTCCCGATGAAAACCAGATTGTGGGACACCAGGTCATTCTGTACCTCTGTCATGGGATGGGCCAGAATCAGTTTTTCGAGTACACGTCCCAGAAAGAAATACGCTATAACACCCACCAGCCTGAGGGCTGCATTGCTGTGGAAGCAGGAATGGATACCCTTATCATGCATCTCTGCGAAGAAACTGCCCCAGAGAATCAGAAGTTCATCTTGCAGGAGGATGGATCTTTATTTCACGAACAGTCCAAGAAATGTGTCCAGGCTGCGAGGAAGGAGTCGAGTGACAGTTTCGTTCCACTCTTACGAGACTGCACCAACTCGGATCATCAGAAATGGTTCTTCAAAGAGCGCATGTTATGA

[0277] T12 LECTIN Amino Acid Sequence:DVTERKQLRDKLQCKDFKWFLETVYPELHVPEDRPGFFGMLQNKGLTDYCFDYNPPDENQIVGHQVILYLCHGMGQNQFFEYTSQKELRYNTHQPEGCIAVEAGMDTLIMHLCEETAPENQKFILQEDGSLFHEQSKKCVQAARKESSDSFVPLLRDCTNSDHQKWFFKERML*

[0278] GalNAc-T13 Lectin Domain:

[0279] The lectin domain polypeptide sequence comprises amino acidresidues 466-639 of GALNT13 (GALNT13 nucleotide sequence accessionnumber is AJ505964).

[0280] T13 LECTIN DNA Sequence:TCTGAGAAGCCAGACTGCATGGAACGCTTGCAGCTGCAAAGGAGACTGGGTTGTCGGACATTCCACTGGTTTCTGGCTAATGTCTACCCTGAGCTGTACCCATCTGAACCCAGGCCCAGTTTCTCTGGAAAGCTCCACAACACTGGACTTGGGCTCTGTGCAGACTGCCAGGCAGAAGGGGACATCCTGGGCTGTCCCATGGTGTTGGCTCCTTGCAGTGACAGCCGGCAGCAACAGTACCTGCAGCACACCAGCAGGAAGGAGATTCACTTTGGCAGCCCACAGCACCTGTGCTTTGCTGTCAGGCAGGAGCAGGTGATTCTTCAGAACTGCACGGAGGAAGGCCTGGCCATCCACCAGCAGCACTGGGACTTCCAGGAGAATGGGATGATTTTTGTACCTGCGTCCGTGTGATGGAAAAGCCCGCCAGCAGTGGCGTTTTGACCAGATCAATGCTGTGGATGAACGATGA

[0281] T13 LECTIN Amino Acid Sequence:EKPDCMERLQLQRRLGCRTFHWFLANVYPELYPSEPRPSFSGKLHNTGLGLCADCQAEGDILGCPMVLAPCSDSRQQQYLQHTSRKEIHFGSPQHLCFAVRQEQVILQNCTEEGLAIHQQHWDFQENGMIVHILSGKCMEAVVQENNKDLYLRPCDGKARQQWRFDQINAVDER*

[0282] GalNAc-T14 Lectin Domain:

[0283] The lectin domain polypeptide sequence comprises amino acidresidues 352-516 of GALNT14 (GALNT14 nucleotide sequence accessionnumber is AJ505991).

[0284] T14 LECTIN DNA Sequence:TATGGAGATGTGTCAGTCAGAAAAACACTAAGAGAAAATCTGAAGTGTAAGCCCTTTTCTTGGTACCTAGAAAACATCTATCCGGACTCCCAGATCCCAAGACGTTATTACTCACTTGGTGAGATAAGAAATGTTGAAACCAATCAGTGTTTAGACAACATGGGCCGCAAGGAAAATGAAAAAGTGGGTATATTCAACTGTCATGGTATGGGAGGAAATCAGGTATTTTCTTACACTGCTGACAAAGAAATCCGAACCGATGACTTGTGCTTGGATGTTTCTAGACTCAATGGACCTGTAATCATGTTAAAATGCCACCATATGAGAGGAAATCAGTTATGGGAATATGATGCTGAGAGACTCACGTTGCGACATGTTAACAGTAACCAATGTCTCGATGAACCTTCTGAAGAAGACAAAATGGTGCCTACAATGCAGGACTGTAGTGGAAGCAGATCCCAACAGTGGCTGCTAAGGAACATGACCTTGGGCACATGA

[0285] T14 LECTIN Amino Acid Sequence:YGDVSVRKTLRENLKCKPFSWYLENIYPDSQIPRRYYSLGEIRNVETNQCLDNMGRKENEKVGIFNCHGMGGNQVFSYTADKEIRTDDLCLDVSRLNGPVIMLKCHHMRGNQLWEYDAERLTLRHVNSNQCLDEPSEEDKMVPTMQDCSG SRSQQWLLRNMTLGT*

[0286] GalNAc-T15 Lectin Domain:

[0287] The lectin domain polypeptide sequence comprises amino acidresidues 382-552 of GALNT15 (GALNT15 nucleotide sequence accessionnumber is AJ505966).

[0288] T15 LECTIN DNA Sequence:TCGGGAATGTTGAGAGCAGATTGGACCTGAGGAAGAATCTGCGCTGCCAGAGCTTCAAGTGGTACCTGGAGAATATCTACCCTGAACTCAGCATCCCCAAGGAGTCCTCCATCCAGAAGGGCAATATCCGACAGAGACAGAAGTGCCTGGAATCTCAAAGGCAGAACAACCAAGAAACCCCAAACCTAAAGTTGAGCCCCTGTGCCAAGGTCAAAGGCGAAGATGCAAAGTCCCAGGTATGGGCCTTCACATACACCCAGAAGATCCTCCAGGAGGAGCTGTGCCTGTCAGTCATCACCTTGTTCCCTGGCGCCCCAGTGGTTCTTGTCCTTTGCAAGAATGGAGATGACCGACAGCAATGGACCAAAACTGGTTCCCACATCGAGCACATAGCATCCCACCTCTGCCTCGATACAGATATGTTCGGTGATGGCACCGAGAACGGCAAGGAAATCGGCGTCAACCCATGTGAGTCCTCACTCATGAGCCAGCACTGGGAC ATGGTGAGTTCTTGAG

[0289] T15 LECTIN Amino Acid Sequence:FGNVESRLDLRKNLRCQSFKWYLENIYPELSIPKESSLQKGNIRQRQKCLESQRQNNQETPNLKLSPCAKVKGEDAKSQVWAFTYTQKILQEELCLSVITLFPGAPVVLVLCKNGDDRQQWTKTGSHIEHIASHLCLDTDMFGDGTENGKEIGVNPCESSLMSQHWDMVSS*

[0290] GalNAc-T16 Lectin Domain:

[0291] The lectin domain polypeptide sequence comprises amino acidresidues 396-558 of GALNT16 (GALNT16 nucleotide sequence accessionnumber is AJ505951).

[0292] T16 LECTIN DNA Sequence:AGTGTGGCTACGCGGATAGAGCAGAGGAAGAAGATGAACTGCAAGTCCTTCCGCTGGTACCTGGAGAACGTCTACCCAGAGCTCACGGTCCCCGTGAAGGAAGCACTCCCCGGCATCATTAAGCAGGGGGTGAACTGCTTAGAATCTCAGGGCCAGAACACAGCTGGTGACTTCCTGCTTGGAATGGGGATCTGCAGAGGGTCTGCCAAGAACCCGCAGCCCGCCCAGGCATGGCTGTTCAGTGACCACCTCATCCAGCAGCAGGGGAAGTGCCTGGCTGCCACCTCCACCTTAATGTCCTCCCCTGGATCCCCAGTCATACTGCAGATGTGCAACCCTAGAGAAGGCAAGCAGAAATGGAGGAGAAAAGGATCTTTCATCCAGCATTCAGTCAGTGGCCTCTGCCTGGAGACAAAGCCTGCCCAGCTGGTGACCAGCAAGTGTCAGGCTGACGCCCAGGCCCAGCAGTGGCAGCTGTTGCCACACACATGA

[0293] T16 LECTIN Amino Acid Sequence:SVATRIEQRKKMNCKSFRWYLENVYPELTVPVKEALPGIIKQGVNCLESQGQNTAGDFLLGMGICRGSAKNPQPAQAWLFSDHLIQQQGKCLAATSTLMSSPGSPVILQMCNPREGKQKWRRKGSFIQHSVSGLCLETKPAQLVTSKCQA DAQAQQWQLLPHT*

[0294] In this Example we have defined minimal sequences of functionallectin domains based on multiple sequence alignments. It is clear thatchanges in the length of sequences used may not affect functionality ofthe lectins. Such changes could constitute, for example, plus or minus10-20 amino acid residues of the GalNAc-transferase sequence at theiramino or carboxy termini. For example, the GalNAc-T1 lectin domain maycomprise 10-20 fewer amino acid residues at its carboxy and/or aminotermini than shown in Table VI's T1 lectin domain sequence; i.e. the T1lectin domain could, for example, stretch from amino acids 403-549 ofthe GALNT1 sequence, or, for example, from amino acids 413-539 of theGALNT1 sequence. Additionally, the GalNAc-T1 lectin domain may comprise10-20 more amino acid residues at its carboxy and/or amino termini thanshown in Table VI's T1 lection domain sequence; i.e. the T1 lectindomain could, for example, stretch from amino acids 383-569 of theGALNT1 sequence, or, for example, from amino acids 373-579 of the GALNT1sequence.

[0295] Sf9 cells were co-transfected with pACGP67-GalNAc-transferasesoluble expression constructs and Baculo-Gold™ DNA (Pharmingen) aspreviously described 27. Briefly, 0.4 μg DNA was mixed with 0.1 μgBaculo-Gold DNA and co-transfected in Sf9 cells in 24-well plates.Ninety-six hours post-transfection recombinant virus was amplified in6-well plates at dilutions of 1: 10 and 1:50. Titer of amplified viruswas estimated by titration in 24-well plates. For large scale productionand purification of recombinant secreted enzymes and lectins theamplified vira were used to infect High Five™ cells grown in serum freemedium (Invitrogen) in upright roller bottles shaking at 140 rpm in 27°C. waterbaths. Recombinant proteins were purified by nickel NiTAchromatography using nickel agarose (Qiagen) as recommended by themanufacturer or by consecutive chromatographies on Amberlite,S-sepharose and Mono-S as previously described²⁸.

Example 8

[0296] Direct binding assay for determination of carbohydratespecificity of polypeptide GalNAc-tranferase lectins using solubleGalNAc-transferase enzymes:

[0297] GalNAc-transferase lectins were previously shown to directGalNAc-glycopeptide substrate specificities of someGalNAc-transferase⁴². The mechanism by which the lectin domains mediatethis specificity is unknown but the finding that the monosaccharideGalNAc selectively inhibits GalNAc-glycopeptide specificity of someisoforms suggested that the lectin domains were involved in aninteraction with the substrate at least partly through theGalNAc-residue. Nevertheless, it has not been possible in the pastdespite many different attempts to demonstrate direct binding of theenzyme protein or fragments hereof to glycopeptides or saccharides⁴². Inthis Example a binding assay using HIS-tagged affinity purified andbiotinylated secreted enzyme was developed. HIS-tagged secreted humanGalNAc-T2 and -T4 were prepared from pAC-GP67-T2-sol and pAC-GP67-T4-solcDNA 8, 26 by PCR as described in Example 7.

[0298] Secreted GalNAc-T2 and -T4 and variant proteins were obtainedfrom infected High Five™ cells crown in serum-free medium (Invitrogen)in upright roller bottles shaken 140 rpm in waterbaths at 27° C.Purification of the recombinant proteins were performed by iminodiaceticacid metal affinity chromatography (IMAC) Ni²⁺-charged (QIAGEN). Elutionwas achieved with 250 mM imidazole in 50 mM sodium phosphate (pH 8.0)and 500 mM NaCl. In some cases, recombinant proteins were purified byconsecutive ionexchange chromatographies as developed and describedpreviously²⁸, before final purification by Ni²⁺-chromatography. Proteinseluted were dialyzed three times against PBS (10 mM sodium phosphate (pH7.4) 150 mM NaCl) and concentrated by centrifugal filter device(Millipore; 10,000 kDa cut off). Purity was analyzed by SDS-PAGE underreducing conditions, and stained for proteins with Coomassie Blue R 250.

[0299] Protein biotinylation was made as previously reported⁶². The pHof 1 ml purified protein (0.3 mg/ml) in PBS was adjusted to pH 9 with 1M NaOH and 40 μl N-hydroxy-succinimidobiotin (Sigma) dissolved in DMF(10 mg/ml) was added. The solution was mixed end-over-end for 2 hours atroom temperature, and dialyzed three times against PBS and an equalvolume of glycerol was added. The biotinylated proteins were stored at−20° C. in 50% glycerol until use.

[0300] Glycosylation of MUCI peptides (0.1 mM) was made in 20 mMcacodylate buffer (pH 8.0), 10 mM MnCl₂, 10 mM UDP-GalNAc, and 20 μgpurified polypeptide GalNAc-T1 or -T2 with or without subsequentglycosylation with GalNAc-T4 at 37° C. during overnight. Glycopeptideswere purified by C-18 reverse phase HPLC. Peptides were customsynthesized by Neosystems (Strasbourg). Biotinylated Helix Pomatialectin (HPA) was from KemEnTec (Denmark). Anti-MUCI HMFG2 monoclonalantibody was a generous gift from Joyce Taylor-Papadimitriou. Anti-MUC1SE5 monoclonal antibody was developed by immunizing Balb/c mice with60-mer MUC1 tandem repeat peptide glycosylated with 5 moles GalNAc perrepeat. Monoclonal antibodies to the lectin domains of human GalNAc-T2and -T4 were developed as previously described⁸⁻⁶³.

[0301] Direct binding ELISA assay was developed as follows: Polystyrenemicrotiter plates (Maxisorb, Nunc, Denmark) were coated with peptides orenzymatically glycosylated glycopeptides in PBS overnight at 4° C.Plates were washed and blocked with 0. 1% Tween20 and 0.2% BSA in PBSfor 1 h at room temperature, followed by incubation with biotinylatedproteins in PBS with 0.05 % Tween20 for 2 h at room temperature. Afterfour washes with PBS, plates were incubated with 1:2,000 dilutionstreptavidin-HRP (Sigma) in PBS for 30 min at room temperature andwashed four times with PBS. Development was performed with 0.5 mg/mlo-phenylenediamine and 0.02% H₂O₂ at room temperature for 15 min, andreaction stopped by adding 100 μl/well of 0.5 N H₂SO₄. Competitiveinhibition assays were done at end-point titers of GalNAc-transferaseproteins with one hour preincubations with inhibitor.

[0302] In initial binding experiments it was determined that secretedGalNAc-T2 and -T4 could bind their peptides substrates in the presenceof 5 mM UDP and Mn⁺⁺ (may be substituted with other divalent cation),whereas no binding was observed in the absence or when 10 mM EDTA wasincluded. This correlates with our findings that GalNAc-T2 can bepurified on an acceptor substrate peptide in the presence of UDP andMn⁺⁺, and can be eluted by removing UDP in EDTA²⁶. This binding ismediated by the catalytic unit of the enzyme, which exposes theacceptor-binding pocket only in the presence of UDP and Mn⁺⁺, predictedby the ordered catalytic reaction.

[0303] In order to selectively evaluate the binding characteristics ofthe lectin domain, assays were carried out in the absence of UDP andMn⁺⁺. Significant binding to GalNAc-peptides was found for bothGalNAc-T2 and -T4 (FIG. 6).

[0304] We have shown that GalNAc-T4 with a single amino acid change inthe lectin domain selectively impairs the GalNAc-glycopeptide substratespecificity of this enzyme⁴². In agreement with this the lectin mutatedenzyme protein did not bind GalNAc-glycopeptides. The binding toGalNAc-glycopeptides were unaffected by Ca++ and EDTA further confirmingthat the catalytic units of the enzyme proteins are not involve inbinding.

[0305] We have shown that GalNAc and GalNAcα-benzyl inhibit theGalNAc-glycopeptide substrate specificity of GalNAc-T4⁴². Lectins andantibodies to carbohydrates usually recognize the anomeric configurationof the sugar structures they bind. However, surprisingly, both GalNAc-T2and -T4 exhibit equal inhibition with GalNAcα-benzyl (Sigma) andGalNAcβ-benzyl (NuRx, Alberta Research Council (FIG. 7)^(70,71). Similarresults were obtained with other aryl derivatives.

[0306] The methods described in this Example utilize recombinantpolypeptide GalNAc-transferases in binding assays and excludes potentialbinding activity through the catalytic unit. It is clear thatrecombinant polypeptide GalNAc-transferases with mutations thatinactivates the binding activity of the catalytic unit can be used, aswell as any truncation of the enzyme protein that eliminate the bindingactivity of the catalytic unit.

[0307] While this binding assay establishes a method for screening forinhibitors of lection mediated binding mediated through the lectins ofhuman GalNAc-T2 and GalNAc-T4, it is clear that the same method withmodifications can be applied to all animal and mammalian polypeptideGalNAc-transferases with a functional lectin domain. The ligand targetused in this Example is a GalNAc-MUC1 glycopeptide producedenzymatically from synthetic peptides. It is clear thatGalNAc-glycopeptides based on any number of peptides with GalNAcattached can be used as target for the binding assay. It is also clearthat the assay developed can be modified to accommodate high throughputscreening by any assay method available in the prior art that can detectand quantify binding between the polypeptide GalNAc-tranferase mediatedthrough its lectin domain and a suitable ligand.

Example 9

[0308] Direct binding assay for determination of carbohydratespecificity of polypeptide GalNAc-tranferase lectins using truncatedGalNAc-transferase lectin domains:

[0309] In Example 8 it was established that secreted solubleenzymatically active proteins of GalNAc-T2 and -T4 bind GalNAc-MUC1glycopeptides, and that GalNAc could inhibit the binding. The catalyticunit of polypeptide GalNAc-transferase can interact and bind acceptorsubstrate peptides and possible glycopeptides⁵², however, bindingstudies without donor substrates (UDP), and in the presence of EDTA tochelate Mn²⁺, suggested that the binding was not mediated through thecatalytic unit. In this Example direct binding to GalNAc-glycopeptidesthrough the lectin domains of polypeptide GalNAc-transferases GalNAc-T2and -T4 was established. Attempts to express C-terminal truncatedGalNAc-transferase proteins failed due to low secretion rate presumablerelated to folding problems and intracellular degradation. Similarphenomenon has recently been reported for GalNAc-T1⁴⁵. Numerous attemptsto express isolated lectin domains in insect cells and P. pastoris havefailed due to low expression and apparent degradation. As described inExample 8 successful expression was finally achieved with constructstruncated as described in Table V using an expression vector with aN-terminal HIS tag and thrombin cleavage site as well as a T7 tag.HIS-tagged truncated GalNAc-T2 and -T4 lectins were expressed andpurified. Lectins were used in binding studies directly or afterbiotinylation as described in Example 8. In binding studies usinglectins without biotinylation antibodies to the HIS-tag and the T7-tag,or in some experiments antibodies raised to GalNAc-T2 and -T4 enzymeswere used to detect binding.

[0310] Inhibition experiments were used to further define the bindingspecificity of GalNAc-T2 and -T4 secreted soluble enzymes as well aslectin domains compared to Helix Pomatia lectin (Table VII). GalNAcα-and GalNAcβ-aryl structures inhibited binding of both enzymes andisolated lectins at comparable levels. In contrast, Helix Pomatia showedstrong preference for GalNAcα-derivatives. Gal and other sugars had noinhibitory effect. Interestingly, UDP-GalNAc was not a significantinhibitor of the GalNAc-transferase lectin binding, but a stronginhibitor of Helix Pomatia binding. Carbohydrates T2ld sT2 T4ld sT4 HPAGlc >100^(a) >100 >100 >100 >100 GlcNAc >100 >100 >100 >100 8BzlαGlcNAc >100 >100 >100 >100 2 BzlβGlcNAc >100 >100 >100 >100 >20Gal >100 >100 >100 50 >100 MeαGal >100 >100 >100 50 >100MeβGal >100 >100 >100 40 >100 GalNAc 37 15 5 1 2 BzlαGalNAc 20 15 5 10.5 PhlαGalNAc 15 5 5 1 1 oNPαGalNAc 12 7 5 0.5 1.5 oNPβGalNAc >12 10 81 >20 pNPαGalNAc >10 >10 10 1 2 pNPβGalNAc >10 >10 8 0.8 >20UDPαGalNAc >100 65 50 30 2 UDP >100 >100 >100 >100 >100Lactose >100 >100 >100 >100 ND EDTA >10 >10 >10 >10 >10

[0311] While this binding assay establishes a method for screening forinhibitors of isolated human GalNAc-T2 and GalNAc-T4 lections, it isclear that the same method with modifications can be applied to allanimal and mammalian polypeptide GalNAc-transferase lectins. The ligandtarget used in this Example is a GalNAc-MUC1 glycopeptide producedenzymatically from synthetic peptides. It is clear thatGalNAc-glycopeptides based on any number of peptides with GalNAcattached can be used as target for the binding assay. It is also clearthat the assay developed can be modified to accommodate high through putscreening by any assay method available in the prior art that can detectand quantify binding between the isolated lectin and a suitable ligand.

[0312] The methods described in this Example utilize recombinantGalNAc-transferase lectins in binding assays which excludes potentialbinding activity through the catalytic unit. It is clear thatrecombinant polypeptide GalNAc-transferases with mutations thatinactivates the binding activity of the catalytic unit can be used, aswell as any truncation of the enzyme protein that eliminate the bindingactivity of the catalytic unit.

Example 10

[0313] Establishment of cell line model systems for cell surfaceexpression of mucin and secreted mucin—Stably transfected CHO and CHOldlD cells:

[0314] Cell Lines and Expression Constructs:

[0315] Wild type Chinese Hamster Ovary cells (CHO) and the glycosylationdeficient mutant cell line CHO ldlD⁵³ were stably transfected with afull coding MUC1 construct (MUC1F, supplied by M. A. Hollingsworth,Neb., USA) containing 32 tandem repeats using the pCDNA3 vector(Invitrogen). A secreted MUC1 construct (MUC1-IgGHIS) was generate byinsertion of mouse IgGγ2a domain fused to 6×histidine tag at the BsU36Isite downstream of the tandem repeat region of MUC1F⁶⁴. Cells weregenerally grown in Hams F12 containing 10% Fetal Bovine Serum at 37° C.at 5% CO₂, and plated 12-24 hours prior to transfection in 6 well platesand grown to approximately 50% confluency. One hour before transfectioncells were washed in serum free medium Optimem (Invitrogen) and cellswere transfected with 1-2 μg DNA using the Lipofectamine plus reagent(Invitrogen) in a total volume of 1 mL as recommended by the supplier.Three hours after the transfection one mL of Hams F12 containing 10%Fetal Bovine Serum was added and cells grown 24-48 hours before mediumwas replaced with 2 mL Hams F12 containing 10% Fetal Bovine Serum. Twoto three days after transfection cells were trypzinized and plated in 75mL T-flasks or in 24/96 well microtiter plates in the same mediumcontaining the appropriate selection agent (1 mg/mL G418 or 0.4 mg/mLZeocin). Selection medium was changed twice weekly until clonesappeared. The medium used for CHO ldlD cells included 1 mM GalNAc and0.1 mM Gal. Transfectant clones were selected by immunocytology withanti-MUC1 antibodies and SDS-PAGE western blot analysis to demonstratecell surface expression and secretion of MUC1.

[0316] Immunocytology

[0317] Two different procedures were applied: i) For general screeningpurposes, cells grown in plates or flasks were trypsinized, washed insaline, and airdried on multiwell coverslides. Slides were fixed inice-cold acetone and stained with monoclonal antibodies andFITC-conjugated rabbit anti-mouse Ig as previously described⁵⁰. ii) Foranalysis of cell surface expression, cells were seeded in 6 well platesand grown for 6 hours in Hams F12 medium with serum until approximately30-50 % subconfluent. Medium was hereafter replaced with Optimemsupplemented with 1.0 mM GalNAc and 0.1 mM Gal and cells grown for 18 to42 hours. Cells were washed once in PBS (phosphate buffered salinewithout Calcium and Magnesium) and subsequently fixed in 2 ml 3%paraformaldehyde at 25° C. for 20 min followed by three washes with PBS.Free aldehyde groups were quenched by incubating in 2 mL 50 mM AmmoniumChloride in PBS for 10 min, followed by three washes with PBS and threewashes with 5 min incubations each with PBS containing 0.2 % Fish SkinGelatin (Sigma). Immunostaining of cells was performed by incubationwith monoclonal antibodies for 40 min at 25° C., followed by threewashes with PBS and three washes of 5 min each with PBS containing 0.2%Fish Skin Gelatin. Subsequently, cells were incubated withFITC-conjugated rabbit anti-mouse Ig (Dako, F261) diluted 1: 150 in PBScontaining 0.2% Fish Skin Gelatin) for 20 min at 25° C., followed by thesame washing procedure, after which wells were cut out of plates andmounted with glycerol as for glass slides.

[0318] Characterization of Wild Type and MUC1 Stable Transfectant CHOCells:

[0319] Several representative clones expressing the full coding orsecreted MUC1 construct were selected and characterized for expressionof MUC1 as well as O-glycosylation. Wild type CHO/MUC1F-clone1 expressedMUC1 at the cell surface as detected by anti-MUC1 monoclonal antibodieson non-permeabilized cells, while wild type CHO/MUC1sol-cloneC4 only waslabeled weakly at the surface. Staining with a panel of anti-MUC1antibodies of permeabilized cells showed intracellular accumulation ofMUC1 detected by HMFG2 (general anti-MUC1 reactive,⁶⁵), SM3 (reactivewith cancer-associated MUC1,⁶⁵), VU-4H5 (reactive with low densityO-glycosylated MUC1, ⁶⁶), VU-2G7 (reactive with high densityO-glycosylated MUC1,⁶⁶), and a novel antibody 5E5 reactive exclusivelywith STn/Tn-glycosylated MUCI glycoforms. In contrast, staining ofnon-permeabilized cells were only reactive with the anti-MUC1 antibodiesHMFG2, SM3 and weakly VU-2G7. Analysis of O-glycosylation using a panelof anti-carbohydrate monoclonal antibodies revealed that wild type CHOcells label very weakly with anti-T antibodies (HH8, 3C9,⁶⁸) at thesurface after neuraminidase treatment, while untreated cells arenegative indicating that wild type CHO cells express very littleO-glycoproteins and the glycosylation is mainly of sialylated core 1structure (ST) (FIG. 8). Antibodies to Tn (1E3, 5F4,⁶⁸) were weaklyreactive without and with neuraminidase treatment and antibodies to STn(TKH2, 3F1,⁶⁸) were negative. Staining with the lectins PNA (T), HPA(Tn), SNA (α2,6sialic acid) and MAA (α2,3sialic acid) were in agreementexcept the finding of weak reactivity with SNA indicating some presenceof α2,6 linked sialic acids which may be derived from N-linked orO-linked glycans. These results demonstrate that the main form ofO-glycosylation found on MUC1 expressed in wild type CHO is the sialyl-Tstructure as found for other recombinant glycoproteins⁶⁹.

[0320] Staining of permeabilized wild type CHO/MUC1sol-clone-C4 withanti-MUC1 antibodies revealed strong intracellular expression of MUC1with HMFG2, SM3, vu-4H5, and vu-2G7 (FIG. 8) Staining withanti-carbohydrate antibodies revealed strong intracellular staining withanti-T after neuraminidase only, while anti-Tn only labeled weakly.These results indicate that the main glycosylation of MUC1 in CHO wildtype is ST similar to untransfected cells.

[0321] In order to characterize the secreted MUC1 product SDS-PAGEwestern blot analysis of harvested culture medium of confluent cultureswere performed. Ten to twenty μL culture supernatant was analyseddirectly or treated with 0.1 U/mL neuraminidase (C. Perfringes VI,Sigma) for 30-60 min at 37° C. Samples were mixed with SDS samplebuffer, reduced with DTT, and run on precast 4-20% gels (Biorad). Asshown in FIG. 9 anti-MUC1 antibodies detected two forms of MUC1 in themedium; a low molecular weight form migrating as 130-140 kdcorresponding to unglycosylated product, and a high molecular weightform migrating above 250 kd. The high molecular weight form wassensitive to neuraminidase treatment as evidenced by a marked shift andretardation in migration. It is known that sialylated glyoproteinsmigrate aberrantly and often desialylation results in slower migrationby SDS-PAGE analysis regardless of the mass. Interestingly, the antibodyVU-4H5 reacted mainly with the unglycosylated form and only a very weakband was found in the high molecular weight forms after neuraminidasetreatment. This result indicates that the PDTR region is O-glycosylatedas the VU-4H5 antibody was previously found to tolerate O-glycosylationmost positions in the tandem repeat except the PDTR region⁶⁶. Inagreement with this the antibody VU-2G7 raised against a MUC1GalNAc-glycopeptide with only one GalNAc per repeat attached in the PDTRregion reacted strongly with the secreted MUC1. Furthermore, reactivitywith the anti-T antibody after neuraminidase treatment showed that themain type of O-glycosylation on secreted MUCI was sialylated-T. Anti-Tnand STn produced no staining.

[0322] Characterization of mutant and MUC1 stable transfectant CHO ldlDcells: CHO ldlD cells stably transfected with full coding MUC1, e.g.CHOldlD/MUC1F-clone2, expressed MUC1 at the cell surface as detected byanti-MUC1 antibodies when cells were grown in GalNAc and Gal (FIG. 10).Cells were seeded at approximately 30-50% confluency (approx. 0.2×10⁶per 6 well plate) in Hams F12 medium supplemented with 10% Fetal Bovineserum and grown for 6 hours. Medium was replaced with Optimem with orwithout 1.0 mM GalNAc and/or 0.1 mM Gal, and cells grown for 18-36 hoursafter which cells were trypsinised and washed in saline and processed asdescribed for immunocytology. CHOldlD/MUC1F-clone2 cells grown in theabsence of sugars and analysed after permeabilization produced verylittle MUC1 detectable by HMFG2 but not by SE5. In contrast, cells grownin the presence of only GalNAc strongly expressed MUC1 as evaluated byHMFG2 and 5E5, specifically reactive with GalNAc-MUC1. In agreement withreactivity with 5E5 these cells also labeled strongly with anti-Tnantibodies, SF4 and 1E3, while anti-T antibodies, HH8 and 3C9, did notlabel the cells. Very weak or no staining with anti-STn antibodies (3F1and TKH2) indicates that α2,6 sialylation to form STn does not occur inCHO ldlD cells. CHOldlD/MUC1F-clone2 cells grown in the presence of bothGalNAc and Gal show reactivity at the surface with anti-T antibodies(HH8 and 3C9) only after neuraminidase pretreatment, confirming resultsthat the predominiant glycoform in CHO cells is sialyl-T (FIG. 10). Nostaining with anti-Tn or STn antibodies was detected with cells grown inboth Gal and GalNAc. CHOldlD/MUClF-clone2 cells grown in the absence ofGalNAc and Gal or in the presence of GalNAc alone showed no reactivitywith anti-Tn and T antibodies or lectins (DBA, HPA, VVA, PNA, not shown)were detected indicating complete lack of O-glycosylation (FIG. 10).Cell surface expression of MUC1 was detected in CHOldlD/MUClF-clone2cells grown in the presence of GalNAc, while cells grown without GalNAcshowed no or only weak expression of MUC1 at the surface (FIG. 10).Surface expression of MUC1 was detected with HMFG2 in cells grown inGalNAc as well as cells grown in both Gal and GalNAc, however,expression analysed with the Tn/STn-MUC1 glycoform specific antibody 5E5revealed surface expression only with cells grown in the presence ofGalNAc (FIG. 10). This latter finding is in agreement with theO-glycosylation pattern determined above in these cells.

[0323] CHOldlD/MUCsol-cloneD5 secretes MUC1 to the culture medium, andpermeabilized cells immunostain with antibodies to MUC1 in thecytoplasm. Cells were grown in Hams F12 medium supplemented with 10%Fetal Bovine serum and seeded at a density of 0.2×10⁶ in 6 well plates.Following growth for 6 hours, the medium was replaced with Optimemsupplemented with 1 mM GalNAc, 0.1 mM Gal, or 1 mM GalNAc and 0.1 mMGal, and cells grown for 18-72 hours. Secretion of MUC1 was monitored byimmunochemical assays of culture supernatants at differing time points.SDS-PAGE western blot analysis was performed with 5 μl culturesupernatant mixed with 5 μl of 2×SDS sample buffer containing 1 mM DTT.Samples were heated to 100° C. for 2 min and loaded on a precast 4-20%gradient gel and run at 125 V for 75 min. Transfer to nitrocellulosemembrane was performed by elecroblotting using Biorad Mini Trans Blotapparatus at 350 mA for 1 hour. Membranes were blocked with 15% skimmedmilk prepared in dH₂O for 2 hours and stained with anti-MUC1 andanti-carbohydrate monoclonal antibodies for 18 hours at 4° C., followedby washing with Tris buffered saline (TBS) (10 mM Tris pH 8,0 with 8.5%NaCl) 5 times for 5 min, and incubation with with biotinylated rabbitanti-mouse IgG subclass specific antibodies (1:1000 dilution in TBS) for1 hour at 25° C. Following 5 washes for 5 min each in TBS, membraneswere incubated in HRP conjugated Streptavidin (1:3000 dilution in TBS)for 30 min at 25° C. After 5 washings of 5 min each in TBS the blot wasdeveloped in 0.04% 4-Chloro-1-Naphthol prepared in 50 mM Tris-HCl (pH7,4) containing 0.025% H₂O₂. Similar to the findings with full codingMUC1 expressed at the cell surface of CHO ldlD cells, glycosylation ofthe secreted MUC1 was dependent on Gal and GalNAc sugars in culturemedium. Cells grown without sugars produced and secreted low amounts ofa low molecular weight MUC1 molecule of apparent mw of 120-130 kdwithout glycosylation detectable by HMFG2 but not 5E5 or anti-Tn andanti-T antibodies (FIG. 11). In contrast, cells grown in 1 mM GalNAcsecreted MUC1 glycosylated with GalNAc (Tn) as evidenced by reactivitywith both HMFG2 and 5E5 as well as anti-Tn antibodies (FIG. 11). Theapparent molecular weight of secreted Tn-MUC1 was 250-300 kd and nosignificant shift in migration was observed with pretreatment withneuraminidase (0.1 U/ml for 30 min at 37° C.), suggesting lack ofα2,6sialylation (STn). This was confirmed by lack of staining withanti-STn antibodies. Cells grown in both 0.1 mM Gal and 1 mM GalNAcproduced and secreted MUC1 with sialylated core 1 (T) glycoformsreactive with HMFG2 but not 5E5 (FIG. 11). Pretreatment withneuraminidase resulted in a significant shift in migration andreactivity with anti-T antibodies as well as the lectin PNA. Two novelanti-MUC1 antibodies described recently have been suggested to reactwith the MUC1 tandem repeat sequence without (Mab VU-4H5) or with (MabVU-2G7) O-glycans attached in the central immunodominant epitope -PDTR-.Analysis of secreted MUC1 produced in CHO ldlD cells grown withoutGalNAc show reactivity with unglycosylated MUC1 migrating at mw 120-130kd with VU-4H5, while no or only weak reactivity was observed when grownin GalNAc with or without Gal (FIG. 12). In contrast, the Mab VU-2G7reacted strongly with MUC1 migrating at 250-300 kd secreted from cellsgrown in GalNAc with or without Gal (FIG. 12). Although, Mab VU-2G7reacted weakly with unglycosylated MUC1 the combined results suggestthat MUCI produced in CHO ldlD cells carry O-glycans on all five sitesof the tandem repeat.

Example 11

[0324] The inhibitor GalNAcα-benzyl inhibits MUC1 expressionindependently of O-glycan processing.

[0325] As shown in Example 10 CHOldlD/MUClF-clone2 cells grown in thepresence of GalNAc but not Gal have limited O-glycosylation capacity,only produce the Tn glycoform of MUC1, but expresses comparable levelsof MUC1 at the cell surface as in wild type CHO cells or in CHO ldlDcells grown in both GalNAc and Gal. This suggested that cell surfaceexpression was not related to O-glycosylation and particular glycoformsas previously proposed (for a review see Huet). We thereforeinvestigated the effect of treatment with GalNAcα-benzyl ofCHOldlD/MUC1F-clone2 cells grown in the presence of GalNAc.CHOldlD/MUC1F-clone2 cells were seeded in 6 well plates at a density of0.2×10⁶ per well and were grown for 6 hours in Hams F12 medium withserum until approximately 30% subconfluent. Medium was hereafterreplaced with Optimem supplemented with 1.0 mM GalNAc or 1.0 mM GalNAcand 0.1 mM Gal with or without the inhibitors GalNAcα-benzyl,GalNAcβ-benzyl or the control GlcNAcα-benzyl. After 18 hours, the mediumwas replaced with fresh Optimem containing the sugars and benzylderivatives as above and grown for 12-48 hours. Initially we analyzedsurface expression of MUC1 by immunocytology. Cells were washed once inPBS-CMF (phosphate buffered saline Calcium and Magnesium free) aftercarefully removing the medium from the wells and subsequently fixed in 2ml 3% paraformaldehyde at 25° C. for 20 min followed by three washeswith PBS-CMF 3 times. Free aldehyde groups were quenched by incubatingin 2 mL 50 mM Ammonium Chloride in PBS-CMF for 10 min, followed by threewashes with PBS-CMF and three washes with 5 min incubations each withPBS-CMF containing 0.2% Fish Skin Gelatin (Sigma). Immunostaining ofcells was performed by incubation with monoclonal antibodies for 40 minat 25° C., followed by three washes with PBS-CMF and three washes of 5min each with PBS-CMF containing 0.2% Fish Skin Gelatin. Subsequently,cells were incubated with FITC-conjugated rabbit anti-mouse Ig (DakoF261) diluted 1: 150 in PBS-CMF containing 0.2 % Fish Skin Gelatin) for20 min at 25° C., followed by the same washing procedure, after whichwells were cut out of plates and mounted with glycerol as for glassslides. FIG. 13 shows that treatment with 1 mM GalNAcα-benzyl, producedstrong inhibition of cell surface expression of MUC1, while treatmentwith a similar control benzyl derivative showed no inhibition.GlcNAcα-benzyl was chosen as a control because this sugar does not serveas a substrate for mammalian glycosyltransferases and hence was notexpected to interfere with O-glycosylation in CHO cells. Most anti-MUC1antibodies reacted with cells grown in GalNAc including VU-2G7 and 5E5,and only VU-4H5 did not react. Reactivity with VU-2G7 indicates that the-PDTR- region is O-glycosylated, while reactivity with 5E5 confirms thatthe glycoforms of surface MUC1 is mainly or exclusively Tn.

[0326] We next analysed the expression of MUCI produced byCHOldlD/MUC1F-clone2 cells by SDS-PAGE western analysis. Cells weregrown for 24 hours or 48 hours in the presence of 1 mM GalNAc or 1 mMGalNAc and 0.1 mM Gal to limit core O-glycosylation toGalNAcα1-O-ser/Thr and Galβ1-3GalNAcα1-O-Ser/Thr, respectively. Cellswere further treated with 2 mM GalNAcα-benzyl, GlcNAcα-benzyl or noinhibitor. Cells were washed and lysed at 24 or 48 hours and the lysatessubjected to immunoprecipitation with monoclonal antibody HMFG2, whichbroadly recognize MUC1 glycoforms. Immunoprecipitates were analysed bySDS-PAGE and western blot using HMFG2 antibody to detect MUC1expression. As shown in FIG. 14 the MUC1 glycoforms at 24 hoursexpressed by cells grown in GalNAc or Gal and GalNAc migrated similarlywith only little high molecular weight forms in both, indicating thatsynthesis of sialylated core 1 O-glycans were time limited. At 48 hours,MUC1 glycoforms migrating as higher molecular weight species wereexpressed more pronounced and selectively by cells grown in Gal andGalNAc. Treatment with GlcNAcα-benzyl produced the same glycoforms atsimilar intensity as cells without treatment. In striking contrast,treatment with GalNAcα-benzyl had significant effect after 48 hours. Asignificant reduction in MUC1 expression was found in cells grown inGalNAc as well as in Gal and GalNAc. In the latter case a significantshift in migration into two bands further confirmed that GalNAcα-benzylalso serves as an inhibitor of O-glycan extension and reducesO-glycosylation to GalNAcα1-O-Ser/Thr. Analysis of the same blots withthe anti-MUC1 antibody 5E5 (FIG. 15) produced essentially the sameresults except that the antibody only labeled the lower migrating bandof the two bands labeled by HMFG2 in cells grown in Gal and GalNAc andtreated with GalNAcα-benzyl. This indicates some heterogeneity inglycosylation.

[0327] These results show for the first time that the effect ofGalNAcα-benzyl on mucin transport and surface expression is independentof its effects on O-glycosylation in striking contrast to the prevailinghypothesis⁴⁶. Because cells grown in the presence of GalNAc do notproduce core 1 (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycosylation,GalNAcα-benzyl cannot serve as a competitive substrate for the core 1β3galactosyltransferase and subsequently for sialyltransferases.GalNAcα-benzyl must therefore exert its function on mucin transport byanother unknown mechanism.

[0328] The in vivo cell line model system developed is one example of amethod to screen for inhibitors effects of one or more compounds ontransport of mucins and O-linked glycoproteins in cells. The Exampleutilizes MUCI but any mucin or O-linked glycoprotein could be used withappropriate expression constructs, antibodies and reagents. Thedeveloped cell model and modifications hereof can be used for highthroughput screens of inhibitors in combination with or as a secondscreen after the binding assays disclosed in Examples 8 and 9.

Example 12

[0329] Identification of a novel selective inhibitor, GalNAcβ-benzyl, ofpolypeptide GalNAc-transferase lectins that inhibits MUC1 expressionwithout affecting O-glycosylation.

[0330] As shown in Examples 8 and 9, polypeptide GalNAc-transferasescontain lectin domains with binding properties for GalNAc-peptidesincluding GalNAc-MUC1 peptides. Since GalNAcα-benzyl was found toinhibit the binding properties of GalNAc-transferase lectins, we testedthe possibility that the independent effect on mucin expression thisO-glycosylation inhibitor has, could be related to an inhibitory effecton GalNAc-transferase lectins. In Examples 8 and 9 we found surprisinglythat the lectin domains of several GalNAc-transferases in addition toGalNAcα-benzyl, which mimics the GalNAc-glycopeptide targets of thelectins, also were inhibited by βGalNAc derivatives. Initial tests withcommercially available GalNAcβ and GalNAcα derivatives, p-nitrophenyland umbrelliferyl did not produce significant effects in our modelsystem. GalNAcβ-benzyl, the β-anomeric configuration of GalNAc-benzyl(there is a β linkage between the N-acetylgalactosamine and the benzylring), was custom synthesized by Alberta Research Council (Canada), andits structure was confirmed by mass spectrometry and ¹H-NMR.CHOldlD/MUC1F-clone2 cells were grown for 12 hours in the presence of 1mM GalNAc or 1 mM GalNAc and 0.1 mM Gal to limit core O-glycosylation toGalNAcα-O-Ser/Thr and Galβ1-3GalNAcα1-O-Ser/Thr, respectively. Cellswere then treated with 2 mM GalNAcα-benzyl, GalNAcβ-benzyl, orGlcNAcα-benzyl as control (GlcNAcα-benzyl was shown in Example 10 tohave no effect). Cells were washed and lysed as described above after 36hours and the lysates subjected to immunoprecipitation with anti-MUC1monoclonal antibodies HMFG2 or 5E5. FIG. 16 illustrates that treatmentwith GalNAcβ-benzyl produced the same or better reduction in MUC1expression as treatment with GalNAcα-benzyl in cells grown in GalNAc aswell as in Gal and GalNAc. In cells grown in Gal and GalNAc MUC1expression was reduced with GalNAcβ-benzyl treatment, but in contrast tocells treated with GalNAcα-benzyl, GalNAcβ-benzyl produced no change inthe migration of MUC1 demonstrating that this inhibitor does not affectthe O-glycan processing. The lack of immunoprecipitation of MUCI byantibody 5E5 in cells grown in Gal and GalNAc indicates that MUC1, isglycosylated with more complex structures than GalNAcα1-O-Ser/Thr asrecognized by this antibody. As shown in FIG. 18 the main O-glycanphenotype of CHO ldlD cells grown in Gal and GalNAc is sialylated-T, and5E5 does not react with MUC1, with T or sialylated T glycoforms of MUC1.FIG. 17 illustrates the same experiments as in FIG. 16 except that thedetection antibody is 5E5 and only Tn and STn MUC1 glycoforms arevisualised. This experiment confirms the strong inhibition of MUC1expression in GalNAcα-benzyl and GalNAcβ-benzyl treated cells.

[0331] The finding that GalNAcα-benzyl and GalNAcβ-benzyl exhibit thesame inhibitory effect on GalNAc-transferase lectin binding, and thatthey have similar effects on inhibition of MUC1 expression, clearlyindicate that the effects these compounds have on mucin expression andsecretion are directed through interaction with the lectin domains ofpolypeptide GalNAc-transferases. The effect GalNAcα-benzyl has onO-glycan processing is a separate phenomenon directed by its ability toserve as a competitive substrate for the core 1⊕1,3galactosyltransferase.

[0332] GalNAcβ-benzyl is the first identified selective inhibitor ofpolypeptide GalNAc-transferase lectins and their roles in transport andsecretion, which does not modulate O-glycosylation in cells (i.e. doesnot serve a substrate for mainly core 1 β3galactosyltransferaseactivities, α2,6sialyltransferase activities, and core 3β3GlcNAc-transferase activities). Inhibitors structurally related toGalNAcβ-benzyl with the same properties, as identifiable by the bindingassays disclosed in Examples 9 and 10, may be designed and syntesized toobtain higher affinity binders. Such inhibitors may be based oncarbohydrates such as the monosaccharide GalNAc or modificationsthereof, inhibitors may be based on structural and functional mimeticssuch as polypeptides, glycopeptides, DNA, RNA, antibodies, and antibodyfragments including phage antibodies, and inhibitors may be natural orsynthetic organic or inorganic compounds. One common feature for suchpreferred inhibitors is the ability to inhibit the binding of one ormore polypeptide GalNAc-transferase lectins to its binding ligand, suchas GalNAc-glycopeptides and mucins as exemplified in Examples 8 and 9.Another feature of the novel inhibitor GalNAcβ-benzyl is its ability toenter living cells and reach the Golgi apparatus for in vivo binding topolypeptide GalNAc-transferase. The hydrophic benzyl aglycone is oneexample of an aryl compound suitable for the β-anomeric configuration ofGalNac-R, but other aryl substituents include, without limitation,p-nitrophenyl umbrelliferyl, and naphtalenmethanol. Any pharmaceuticalcarrier known in the art may be used to achieve the same effect. Theappropriate carrier will be evident to those skilled in the art and willdepend in large part upon the route of administration.

Example 13

[0333] Inhibition of Secretion of Mucins:

[0334] Because GalNAcα-benzyl exerts separate effects on O-glycanprocessing and mucin expression, the use of the novel selectiveinhibitor of mucin expression, GalNAcβ-benzyl, allow analysis of mucinexpression and secretion in different cell line models. Examples ofhuman cell lines (available from ATCC, USA) expressing and secretingmucins are without limitations LS174T, HT29, Colo205, CALU, MCF7, T47D,NCI-H292, and A549. Most human adenocarcinoma cell lines express andsecret mucins and analysis with antibodies to detect protein expressionor probes to detect mRNA can reveal the types and quantities of mucins.The human colon carcinoma cell line LS174T was previously shown toexhibit reduced secretion of mucin following treatment withGalNAcα-benzyl²⁰. This and other cell lines can be used to treat with 2mM GalNAcβ-benzyl. In this Example we used wild type CHO/MUC1sol-cloneC4and western blot analysis of medium of cells treated with 1-2 mMGalNAcα-benzyl, GalNAcβ-benzyl, or the control GlcNAcα-benzyl. Treatmentwith both GalNAcα-benzyl and GalNAcβ-benzyl showed inhibition ofsecreted MUC1 compared to control treated or non-treated cells. Theinhibitory effect on mucin secretion can be quantified by a number ofassays known to the skilled in the art including western blot, ELISA,gelfiltration, immunocapture, and other assays.

[0335] The in vivo cell line model system developed is one example of amethod to screen for inhibitors effects of one or more compounds onsecretion of mucins and O-linked glycoproteins in cells. The Exampleutilizes MUC1 but any mucin or O-linked glycoprotein could be used withappropriate expression constructs, antibodies and reagents. Thedeveloped cell model and modifications hereof can be used for highthroughput screens of inhibitors in combination with or as a secondscreen after the binding assays disclosed in Examples 8 and 9.

Example 14

[0336] Synthesis of benzyl-2-acetamido-2-deoxy-β-D-galactopyranosidegalactosamine (GalNAcβ-benzyl)

[0337] The synthesis of benzyl-2-acetamido-2-deoxy-β-D-galactopyranosidegalactosamine (GalNAcβ-benzyl) was carried out by usingGalactosamine-hydrochloride as starting material. Treatment of thiscompound with Troc reagent provided the N-Troc derivative. The reactionwas carried out in aqueous sodium hydrogen carbonate solution for 15hours at ambient temperature. After evaporation and co-evaporation ofthe mixture with toluene, the crude mass was subjected to acetylationwith acetic anhydride and pyridine to obtain the peracetylatedderivative of the N-Troc-galactosamine. The acetylated product wasconverted into its thiobenzyl glycoside by using boronrifluorideethereate solution as a catalyst. The N-Troc derivative was convertedinto its corresponding azido compound by reacting the compound with atriflic azide and copper sulfate. The2-azido-2-deoxy-3,4,6-tri-O-acetyl-β-D-thiogalactoside was thentransformed into bromide derivative by treatment with N-bromosuccinamidein dichlorormethane. The reaction of glycosyl benzyl alcohol in thepresence of bromide with silver carbonate and silver trifluoromethanesulfonate in dichlorormethane provided quantitative yield of β-benzylglycoside. After purification on a silica gel column, this product wasdeactylated with sodium methoxide and methanol. Azido group of thecompound was reduced with H2S in the presence of triethylamine-pyridineand water solution to provide 2-amino galactoside, which wasN-acetylated with acetic anhydride and methanol solution containingsodium hydrogen carbonate. Theβ-O-benzyl-2-deoxyl-2-acetamido-galactopyranoside compound was finallypurified to homogeneity by chromatography on silica gel.

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We claim:
 1. A method of modulating functions of polypeptideGalNAc-transferases comprising administering an effective amount of anappropriate agent which is effective in modulating functions of one ormore polypeptide GalNAc-transferases.
 2. A method according to claim 1comprising administering an effective amount of an appropriate agentwhich is effective in inhibiting one or more lectin domains ofpolypeptide GalNAc-transferases and modulating functions mediated bysaid lectin domains.
 3. The method of claim 1 where the inhibitedfunction is GalNAc-glycopeptide specificity of polypeptideGalNAc-transferases.
 4. The method of claim 1 where the inhibitedfunction is Galβ1-3GalNAc-glycopeptide specificity of polypeptideGalNAc-transferases.
 5. The method of claim 1 where the inhibitedfunction is UDP-Gal donor substrate specificity.
 6. The method of claim1 wherein said agent is selected from the group consisting ofcarbohydrates, peptides, glycopeptides, glycoconjugates and portions andfragments thereof.
 7. The method of claim 6 wherein said carbohydrate orcarbohydrate portion comprises a GalNAc residue.
 8. The method of claim6 wherein said carbohydrate or carbohydrate portion comprises a Galresidue.
 9. The method of claim 6 wherein said carbohydrate orcarbohydrate portion comprises the Galβ1-3GalNAc disaccharide.
 10. Themethod of claim 6 wherein said carbohydrate or carbohydrate portion islinked to an acceptable carrier.
 11. The method of claim 10 wherein saidcarrier is a benzyl group.
 12. The method of claim 6 wherein saidglycopeptide comprises GalNAc-glycosylated glycopeptides.
 13. The methodof claim 6 wherein said glycopeptide comprisesGalβ1-3GalNAc-glycosylated glycopeptides.
 14. The method of claim 6wherein said peptide represents sequences contained in the tandemrepeats of human and animal mucins.
 15. The method of claim 1 whereinsaid agent is linked to a pharmaceutical carrier.
 16. The method ofclaim 1 wherein the polypeptide GalNAc-transferase is GalNAc-T4.
 17. Themethod of claim 1 wherein the polypeptide GalNAc-transferase isGalNAc-T7.
 18. The method of claim 1 wherein the polypeptideGalNAc-transferase is GalNAc-T2.
 19. The method of claim 1 wherein thepolypeptide GalNAc-transferase is GalNAc-T3.
 20. The method of claim 2wherein the lectin mediated function is glycopeptide specificity ofpolypeptide GalNAc-transferases.
 21. The method of claim 2 wherein thelectin mediated function is peptide specificity of polypeptideGalNAc-transferases.
 22. A method of screening one or more testsubstances for the ability to modulate polypeptide GalNAc-transferaseenzymatic activity in a cell-free or cell-based assay, which comprises:(i) contacting a polypeptide GalNAc-transferase, or a cell thatrecombinantly expresses a polypeptide GalNAc-transferase, with one ormore test substances under assay conditions suitable for the detectionof said enzymatic activity; and (ii) measuring whether said enzymaticactivity is thereby modulated by one or more of the test substances. 23.A method as defined in claim 22, wherein one or more test substances areselected from a combinatorial chemical library.
 24. A method as definedin claim 22, wherein one or more test substances are generated bymethods of polypeptide GalNAc-transferase structure-based design.
 25. Apharmaceutical composition comprising an agent which is effective inmodulating functions of one or more polypeptide GalNAc-transferases, anda pharmaceutically acceptable carrier.
 26. A pharmaceutical compositionaccording to claim 25 wherein said agent is an agent which is effectivein inhibiting one or more lectin domains of polypeptideGalNAc-transferases and modulating functions mediated by said lectindomains.
 27. A pharmaceutical composition according to claim 25 whereinsaid agent is selected from the group consisting of carbohydrates,peptides, glycopeptides, glycoconjugates and portions and fragmentsthereof.
 28. Use of an agent which is effective in inhibiting one ormore lectin domains of polypeptide GalNAc-transferases and modulatingfunctions mediated by said lectin domains for preparing a medicament forthe treatment of tumors and cancers.
 29. Use of an agent which iseffective in inhibiting one or more lectin domains of polypeptideGalNAc-transferases and modulating functions mediated by said lectindomains for preparing a medicament for the treatment of lung diseasesassociated with mucous accumulation.
 30. Use according to claim 29wherein said lung diseases are selected from the group consisting ofasthma, chronic bronchitis, smoker's lung, and cystic fibrosis.
 31. Useof an agent which is effective in inhibiting one or more lectin domainsof polypeptide GalNAc-transferases and modulating functions mediated bysaid lectin domains for preparing a medicament for the treatment ofdiseases of exocrine glands associated with increased or decreased mucinsecretion.
 32. Use according to claim 31 wherein said diseases ofexocrine glands are selected from the group consisting of Sjøgren'ssyndrome and dry mouth.
 33. Use of an agent which is effective ininhibiting one or more lectin domains of polypeptide GalNAc-transferasesand modulating functions mediated by said lectin domains for preparing amedicament for the treatment of disorders associated with dysregulationof selectin-mediated leukocyte trafficking.
 34. Use according to claim33 wherein said disorders associated with dysregulation ofselectin-mediated leukocyte trafficking are selected from the groupconsisting of autoimmunity, arthritis, leukemias, lymphomas,immunosuppression, sepsis, wound healing, acute and chronicinflammation.
 35. An isolated nucleic acid molecule comprising a nucleicacid sequence encoding a polypeptide selected from the group consistingof the lectin domain of a mammalian polypeptide GalNAc-transferase, anda lectin-functional variant or fragment of said lectin domain, whereinsaid polypeptide does not encompass the intact, functioning catalyticdomain of the enzyme.
 36. A nucleic acid molecule according to claim 35comprising a nucleic acid sequence selected from the group consisting ofthe nucleic acid sequences encoding the GalNAc-T1 to -T16 lectin domainsset forth in Table III and lectin-functional variants and fragmentsthereof.
 37. An isolated lectin polypeptide comprising the lectin domainof a mammalian polypeptide GalNAc-transferase or a lectin-functionalvariant or fragment thereof.
 38. A lectin polypeptide according to claim37 having an amino acid sequence selected from the group consisting ofthe amino acid sequences of GalNAc-T1 to -T16 set forth in Table III andlectin-functional variants and fragments thereof.
 39. A method ofproducing a lectin polypeptide comprising the lectin domain of amammalian polypeptide GalNAc-transferase or a lectin-functional variantor fragment thereof, said polypeptide no encompassing the intact,functional catalytic domain of said transfearse, the method comprising:(i) growing a host cell transfected with a nucleic acid sequenceencoding the lectin domain of a mammalian polypeptide GalNAc-transferaseor a lectin-functional variant or fragment of said lectin domain andexcluding the intact catalytic domain of the enzyme under conditionssuitable for lectin expression; and (ii) isolating the lectinpolypeptide produced by the host cell
 40. A method according to claim 39wherein said nucleic acid sequence is selected from the group consistingof the sequences encoding the GalNAc-T1 to -T16 lectin domains stated inTable III herein and lectin-functional variants and fragments thereof.41. A method of identifying a substance that binds to a polypeptideGalNAc-transferase lectin domain, which comprises (i) reacting a lectinpolypeptide according to any one of claims 37, 38 or 55 with at leastone substance which potentially may bind to the polypeptide, underconditions which permit the association between the substance and thepolypeptide; (ii) removing and/or detecting the polypeptide withassociated substance which, if present, indicates that the substancebinds to the polypeptide.
 42. A method of screening for inhibitors offunctions mediated by polypeptide GalNAc-transferase lectin domainswhich comprises using a lectin polypeptide according to any one ofclaims 37 or 38 in a binding assay where it interacts with a GalNAc orGalβ1-3GalNAc O-glycopeptide ligand or a molecular mimic hereof, andmeasuring the binding inhibition to identify and evaluate efficiency ofa potential inhibitor.
 43. A method of screening for inhibitors offunctions mediated by polypeptide GalNAc-transferase lectin domainswhich comprises using a polypeptide GalNAc-transferase or a fragmentthereof retaining functional lectin binding in a binding assay where itinteracts with a GalNAc or Galβ1-3GalNAc O-glycopeptide ligand or amolecular mimic hereof, while the binding capacity of the catalyticdomain is inactivated by the presence of EDTA or the absence of UDP orUDP-GalNAc or Mn⁺⁺ or other divalent metal ion, and measuring thebinding inhibition to identify and evaluate efficiency of a potentialinhibitor.
 44. A compound that binds to the lectin domain of a member ofthe mammalian family of polypeptide GalNAc-transferases and inhibits thebinding of a carbohydrate to said domain, wherein said compound does notserve as a substrate for core 1 β1,3-galactosyltransferase activity orother glycosyltransferases acting in mucin O-glycosylation.
 45. Aninhibitor of polypeptide GalNAc-transferase lectin-mediated functionsthat selectively binds to the lectin domain of said transferase and doesnot serve as an acceptor substrate for core 1 β3-galactosyltransferaseor other glycosyltransferases functioning in O-glycosylation.
 46. Aninhibitor according to claim 45, which is GalNAcβ1-R.
 47. A method ofinhibiting mucin secretion in a subject comprising administering aneffective amount of a compound that binds to one or more lectin domainsof members of a mammalian family of polypeptide GalNAc-transferases andinhibit binding of such domains to carbohydrates.
 48. A method ofinhibiting hypersecretion and accumulation of mucin in the lungs of amammal suffering from a chronic obstructive respiratory pulmonarydisease comprising administering to said mammal an effective amount ofat least one agent that inhibits the binding of polypeptideGalNAc-transferase lectin domains to GalNAc-glycopeptides, wherein saidagent is selected from the group consisting of GalNAcβ1-benzyl, acarbohydrate portion of GalNAcβ1-benzyl, a glycoconjugate that includesa carbohydrate portion of GalNAcβ1-benzyl or a derivative of either thatinhibits the binding of GalNAc-glycopeptides to a GalNAc-transferaselectin domain.
 49. The method of claim 48 wherein the agent is aglycoconjugate that includes a carbohydrate portion of GalNAcβ-benzyl.50. A method of inhibiting the secretion of mucin in a patientcomprising administering to the patient a therapeutically effectiveamount of an agent selected from the group consisting ofGalNAcβ1-benzyl, a carbohydrate portion of GalNAcβ1-benzyl, aglycoconjugate that includes a carbohydrate portion of GalNAcβ1-benzylor a derivative of either that inhibits the binding ofGalNAc-glycopeptides to a GalNAc-transferase lectin domain.
 51. Themethod of claim 50 wherein the patient has a disease selected from thegroup consisting of chronic obstructive pulmonary diseases, asthma, andcystic fibrosis.
 52. The method of claim 50, which selectively inhibitsone or more members of the GalNAc-transferase family without inhibitingother glycosyltransferases selected from the group consisting of core 1β1,3-galactosyltransferases, α2,6-sialyltransferases, andglycosyltransferases functioning in the O-glycosylation pathway.
 53. Thenucleic acid of claim 35 wherein the polypeptide GalNAc-transferase orlectin-functional variant or fragment of said lectin domain is human.54. The polypeptide of claim 37 wherein the polypeptideGalNAc-transferase or a lectin-functional variant or fragment thereof ishuman.
 55. The method of claim 39 wherein the wherein the polypeptideGalNAc-transferase or lectin-functional variant or fragment of saidlectin domain is human.
 56. The compound of claim 44 wherein said saidfamily of polyepeptide GalNAc-transferases is human.
 57. The method ofclaim 48 wherein said mammal is a human.
 58. An inhibitor according toclaim 46 wherein R represents an aglycone.
 59. An inhibitor according toclaim 46 wherein R represents an aryl group.
 60. An inhibitor accordingto claim 46 wherin R is selected from the consisting of benzyl, phenyl,p-nitrophenyl, umbrelliferyl, and naphtalenmethanol.
 61. The nucleicacid of claim 36 further comprising 30-60 nucleotides of thecorresponding GalNAc-transferase sequence at its 5′ or 3′ end.
 62. Thepolypeptide of claim 38 further comprising 10-20 amino acid residues ofthe the corresponding GalNAc-transferase sequence at its carboxy oramino terminus.
 63. A method of modulating the function of one or morelectin domains of a polypeptide GalNAc-transferase comprisingadministering an effective amount of GalNAc 1-R which is effective inmodulating functions mediated by said lectin domains.
 64. The method ofclaim 63 wherein R represents an aglycone.
 65. The method of claim 63wherein R represents an aryl group.
 66. The method of claim 64 where Ris selected from the consisting of benzyl, phenyl, p-nitrophenyl,umbrelliferyl, and naphtalenmethanol.
 67. A method of screening one ormore test substances for the ability to inhibit or modulateintracellular transport and/or cell surface expression of illiucins,O-glycosylated glycoproteins, glycoproteins and proteins in a cell-basedassay, which comprises: (i) contacting a cell that expresses mucins,O-glycosylated glycoproteins, glycoproteins and proteins, with one ormore test substances under assay conditions suitable for the detectionof inhibition or modulation of said expression; and (ii) measuringwhether intracellular transport and cell surface expression of saidmucins, O-glycosylated glycoproteins, glycoproteins and proteins arethereby inhibited or modulated by one or more of the substances.
 68. Amethod of screening one or more test substances for the ability toinhibit or modulate secretions of mucins, O-glycosylated glycoproteins,glycoproteins and proteins in a cell-based assay, which comprises: (i)contacting a cell that secretes mucins, O-glycosylated glycoproteins,glycoproteins with one or more test substances under assay conditionssuitable for the detection of inhibition or modulation of saidsecretion; and (ii) measuring whether secretion of said mucins,O-glycosylated glycoproteins, glycoproteins and proteins are therebyinhibited or modulated by one or more of the substances.